Recent years have witnessed significant progress towards understanding the molecular mechanism of nucleotide excision repair in living cells. Biochemical studies in Escherichia coli, and genetic and molecular studies in lower and higher eukaryotes have revealed an unexpected complexity suggesting interesting protein-protein and protein-DNA interactions. This review considers selected aspects of nucleotide excision repair in E. coli, Saccharomyces cerevisiae and mammalian cells, with a particular emphasis on new observations and on models that may provide explanations for the complexity evident from genetic and biochemical studies.

The concept of excision repair of DNA evolved during the mid-1960s (see Friedberg, 1985, for a review) following the demonstration that when Escherichia coli cells are ultraviolet (u.v.)-irradiated and then incubated, pyrimidine dimers are lost from acid-precipitable (high molecular weight) DNA and can be recovered as acid-soluble material. The additional demonstration of repair synthesis of DNA at about the same time led to a general model for a multistep process in E. coli consisting of the following sequential events:

(1) DNA incision 5′ to sites of base damage

(2) Concomitant 5′ →3′ excision-resynthesis catalysed by DNA polymerase I

(3) DNA ligation.

Mutants of E. coli defective in the excision of pyrimidine dimers are also abnormally sensitive to killing by a variety of chemicals known to damage DNA. This suggested that the model presented above was distinguished by its generality, i.e. that living cells probably excise all forms of base damage by a single molecular mechanism. Progress during the past 15 years has proven this concept to be simplistic. The discovery by Tomas Lindahl and his colleagues in the early 1970s of a repair-specific class of enzymes called DNA glycosylases (see Friedberg, 1985, for a review), was of fundamental importance, for it provided definitive evidence for a distinct and novel mechanism for the excision of damaged and inappropriate (e.g. uracil) bases, involving their excision as free bases. Hence, the phrase base excision repair was coined to distinguish this mode of excision repair of DNA from so-called nucleotide excision repair in which damaged bases are excised as part of a nucleotide structure. Since then, at least one other distinct mode of excision repair has been discovered. In E, coli the process of post-replicative mismatch correction involves the recognition of hemimethylated GATC sequences and of mismatched bases in newly replicated DNA (see Friedberg, 1985, for a review). A cell-free system for mismatch correction has been established (Lu et al. 1983) and although the detailed mechanism of this process has not been elucidated, it is evident that it requires a number of gene products not utilized in either base excision or nucleotide excision repair. Finally, in at least two biological systems, elements of both base and nucleotide excision repair are represented in a ‘hybrid’ biochemical mechanism specific for the removal of pyrimidine dimers from DNA. Thus, in phage T4-infected coli and in Microccus luteus, pyrimidine dimers are excised as part of an oligonucleotide, as is true of nucleotide excision repair (see Friedberg, 1985, for a review). However, the mechanism of DNA incision involves a specific DNA glycosylase and in this respect resembles other examples of base excision repair. It is of ironic historical interest that for many years the enzyme in T4-infected E. coli and M. luteus served as a general model for the study of nucleotide excision repair before it was appreciated that the mechanism of excision repair involving this enzyme is specific for pyrimidine dimers and is apparently limited to these two organisms.

It is now apparent that the general term excision repair of DNA is an oversimplification that embraces multiple diverse biochemical mechanisms. Living cells utilize discrete processes of base excision repair, nucleotide excision repair or mismatch correction, depending on the type of damage present in DNA, and manifest other cellular responses to DNA damage depending on the proximity of damaged bases to sites of active DNA replication. Both excision repair and other cellular responses to DNA damage have been subjects of a number of recent reviews (see Friedberg, 1985, and references therein; Walker, 1985; Walker et al. 1985; Cleaver, 1986; Sedgwick, 1986; Weiss & Grossman, 1986). This review deals exclusively with the process of nucleotide excision repair, with particular emphasis on recent advances in E. coli, the yeast Saccharomyces cerevisiae, and mammalian cells.

ESCHERICHIA COLI: A GENERAL MODEL FOR NUCLEOTIDE EXCISION REPAIR?

The demonstration that mutants in the uvrA, uvrB or uvrC genes of E. coli are defective in the excision of pyrimidine dimers during post-ultraviolet incubation led to a search for the proteins encoded by these genes. These were first partially purified by Seeberg and his colleagues using an assay in which ATP-dependent incision of ultraviolet (u.v.)-irradiated DNA was complemented in extracts of a particular uvr mutant by fractions from uvr+ cells or from different uvr mutants (Seeberg, 1978, 1981; Seeberg et al. 1978). This was an arduous task since constitutive untransformed E. coli cells contain very small amounts of UvrA, UvrB and UvrC proteins (Sancaret al. 1981a,6; Yoakum & Grossman, 1981). Subsequent isolation of the relevant uvr genes by molecular cloning facilitated studies on the genes themselves, as well as their overexpression to yield large amounts of proteins for biochemical studies.

The uvr genes of E. coli

Regulation. While the isolation and overexpression of the uvrA, uvrB, uvrC and uvrD genes from recombinant plasmids have greatly facilitated inroads into the biochemical mechanism of nucleotide excision repair in E. coli (see below), much remains to be learned about the expression of the chromosomal genes, particularly those that are controlled by other regulatory loci. The expression of the uvrA and uvrB genes of E. coli is enhanced fivefold following exposure of cells to DNA- damaging agents, and there is good evidence that the induced expression of these genes is part of the general SOS phenomenon regulated by the recA/lexA system (see Friedberg, 1985, for a review). The uvrA gene contains a single typical prokaryotic promoter with a Pribnow box and a ‘-35 sequence’ appropriately located with respect to a single transcriptional start-site. This promoter is overlapped at its 5’ end by a consensus SOS box and binding of LexA protein to this operator region has been demonstrated in vitro (Sancar et al. 1982b).

Regulation of the uvrB gene is less straightforward. Sequence analysis has identified three distinct promoter regions and three transcriptional start-sites have been mapped by studies in vitro (Sancaret al. 1982a). Two of these promoters (Pl and P2) are located relatively close to each other and to the presumed translation start-site; the third (P3) is located about 300base-pairs (bp) upstream from them. A typical SOS box overlaps P2 (the more distal of the two closely spaced promoters), and in the presence of LexA protein in vitro transcription from this promoter is blocked while that from the more proximal promoter Pl is unaffected (Sancar et al. 1982a). This suggests that constitutive expression of uvrB is from Pl, and that induced expression is from P2 and is regulated by the recA/lexA system. On the other hand, studies in living cells show that both promoters are active in the constitutive and induced states, but Pl is the stronger of the two (van den Berg et al. 1983). Thus, it is possible that all transcription of uvrB is controlled by Pl and that LexA protein regulates expression from Pl by binding to P2. The role, if any, of the most upstream promoter P3 is unknown; however, there are indications that this promoter may be regulated by E. coli DnaA protein (see below).

The situation with respect to the uvrC gene is even more complicated. This gene was not among the many A. coli din (Jamage zwducible) genes isolated by the operon fusion strategy that used the Mud (ApRlac) system (Kenyon & Walker, 1980). Using uvrC-galK fusion plasmids, recA/ZexA-dependent induction of galactokinase has been demonstrated (van Sluis et al. 1983). However, in contrast to other SOS genes, induction of uvrC under these experimental conditions is very slow and is not effected by treatment of cells with nalidixic acid, characteristically a strong inducer of SOS genes. The uvrC gene has been cloned and studied in a number of laboratories; however, the promoter(s) for constitutive expression has not been unambiguously identified. Current evidence suggests the presence of multiple promoters of different strength, and of transcriptional regulation by attenuation (Forster & Strike, 1985). In a recent study two different uvrC fusions to the E. coli cat gene (one of which contains as much as 1200 bp of uvrC upstream sequence) were tested for inducible expression of chloramphenicol acetyl-transferase (CAT). No enhanced expression of CAT was observed in response to treatment with either u.v. radiation or mitomycin C, and the levels of CAT expression were unaffected by mutations in recA or lexA known to prevent induction or to result in constitutive induction of SOS genes (Forster & Strike, 1985). Thus, it would appear that the uvrC gene is not induced by DNA-damaging agents.

There is compelling evidence from cellular-biological studies for involvement of the uvrD gene in nucleotide excision repair in E. coli. Unlike uvrA, uvrB and uvrC strains in uvrD mutants, excision of pyrimidine dimers is not completely abolished. However, such mutants show a reduced rate and extent of loss of thymine-containing pyrimidine dimers during post-irradiation incubation (see Friedberg, 1985, for a review). In addition, the extent (but not the initial rate) of ATP-dependent DNA incision in semipermeabilized u.v.-irradiated cells is reduced in uvrD mutants relative to uvr+ cells (Ben-Ishai & Sharon, 1981). There is also good evidence that the uvrD gene is inducible and under SOS regulation. MrD::Mud (ApRlac) insertion mutants have been identified and yield enhanced /3-galactosidase activity following treatments known to induce SOS genes. Furthermore, the genetics of this response is consistent with recA/lexA regulation (Maples & Kushner, 1982; Siegel, 1983; Kumura & Sekiguchi, 1984). In addition, sequence analysis of the uvrD gene revealed a typical LexA binding box, which functions as an operator for purified LexA protein in vitro and is situated downstream from a single promoter (Easton & Kushner, 1983). In the absence of LexA protein a significant fraction of the mRNA transcribed from this promoter in vitro terminates after about 60 nucleotides at a sequence that resembles a rho-independent terminator (Easton & Kushner, 1983). Hence, like the uvrC gene, expression of uvrD may be subject to regulation by transcriptional attenuation.

Structural features

The uvr and polA genes have been sequenced (Joyce et al. 1982; Finch & Emmerson, 1984; Sanear et al. 1984; Arikan et al. 1986; Backendorf et al. 1986; Husain et al. 1986) and examination of the predicted amino acid sequences of the proteins has revealed a number of interesting homologies. For example, a number of nucleotide binding proteins have regions of amino acid sequence homology that include the tripeptide GKS(T) (single-letter code for amino acids) (Walker et al. 1982). (The third position of the tripeptide is sometimes G, which has a hydropathic index very similar to that of S and T (Kyte & Doolittle, 1982).) Some of the proteins that contain the GKS (T or G) sequence are also purine nucleoside triphosphatases and include the E, coli RecA, UvrD (Finch & Emmerson, 1984) and UvrA (Husain et al. 1986) proteins. A related sequence is also present in UvrB protein (Arikan et al. 1986; Backendorf et al. 1986) as well as in UvrC and DNA polymerase I (Fig. 1). Since the latter protein binds deoxynucleoside triphosphates it can be considered a nucleotide binding protein. Neither triphosphatase nor nucleotide binding activities have been associated with UvrB or UvrC protein; however, a mixture of UvrA and UvrB protein has more ATPase activity than UvrA protein alone (see below), suggesting the possible activation of such an activity in UvrB protein. The sequence of UvrA protein shows two GKS (T or G) regions and examination of their predicted secondary structure shows a striking similarity between them and to other known ATPases, suggesting the presence of two nucleotide binding sites in the protein (Husain et al. 1986).

Fig. 1.

Sequence homology between the predicted amino acid sequences of regions of E. coli and yeast nucleotide excision repair genes. The single-letter amino acid code is used throughout. Boxes denote identical amino acids or amino acids with similar hydropathic values.

Fig. 1.

Sequence homology between the predicted amino acid sequences of regions of E. coli and yeast nucleotide excision repair genes. The single-letter amino acid code is used throughout. Boxes denote identical amino acids or amino acids with similar hydropathic values.

A comparison of the amino acid sequences of the predicted UvrB and UvrC polypeptides shows two other regions of homology (Backendorf et al. 1986; Husain et al. 1986) and it is perhaps relevant in this regard that antisera to UvrB and to UvrC proteins crossreact with both proteins (Backendorf et al. 1986). A region near the amino-terminal end of UvrB protein also shows homology with a region of the predicted amino acid sequence of the alkA gene product, a DNA glycosylase called 3-methyladenine-DNA glycosylase II.

Are uvr genes involved in other DNA transactions?

The study of the E. coli uvr genes has perhaps led to the implicit (if not explicit) assumption that the process of nucleotide excision repair defines their exclusive role in DNA metabolism. This section examines evidence that some of these genes may be involved in other DNA transactions. I particularly wish to address the possibility that in E. coli DNA repair may be linked to processes such as DNA replication, through one or more gene products common to both events. If such linked functions do indeed exist for particular uvr genes, they could have interesting and important regulatory potential. Thus, for example, the sequestration of Uvr proteins by an excision repair complex could limit the amount of these proteins available for a replication complex, hence providing a mechanism for regulating the initiation and/or progression of semi-conservative DNA synthesis during nucleotide excision repair.

Several provocative examples of the possible involvement of uvr genes in DNA replication have been documented. Early studies demonstrated that uvrD mutants that are also defective in thepoZA gene are inviable (Horiuchi & Nagata, 1973; Siegel, 1973; Smirnov et al. 1973), implicating the uvrD gene in DNA replication. More recently, it has been shown that the uvrD gene encodes an ATP-dependent DNA helicase called DNA helicase II (Arthur et al. 1982; Maples & Kushner, 1982; Kumura & Sekiguchi, 1984). Antiserum raised against purified DNA helicase II inhibits the replication of phage A or ColEl DNA in subcellular systems (Klinkert et al. 1980); and the addition of DNA helicase II to a cell-free system stimulates the replication of artificially forked  DNA (Kuhn & Abdel-Monem, 1982). Mutants defective in just the uvrD gene are viable; hence DNA helicase II is apparently not absolutely required for DNA replication. Nonetheless, the results just summarized are consistent with an involvement of UvrD protein in normal DNA replication in E. coli.

The double mutant uvrBpolA is also inviable; an observation that dates back to the early 1970s (Shizuya & Dykhuizen, 1972; Morimyo & Shimazu, 1976). More recently, evidence has emerged for the possible regulation of the uvrB gene by E. coli DnaA protein. DnaA protein is a polypeptide of Mr ≈48×103 required for the initiation of bidirectional replication at oriC, the E. coli origin of replication (Kornberg, 1980). Studies on the specific cooperative binding of DnaA protein to oriC have defined a minimal binding sequence with the consensus (Fuller et al. 1984). This sequence is highly conserved in the chromosomal replication origin of other prokaryotes, in the replication origin of a number of plasmids, and in the upstream regulatory region of a number of E. coli genes, including the dnaA gene itself, pyrBl, argF, polA, dam and uvrB (Fuller et al. 1984). In the case of the dnaA gene, DnaA protein appears to act as a negative auto-regulator of expression. Thus, in cells transformed with dnaA-lacZ fusion plasmids, the expression of β-galactosidase correlates inversely with levels of DnaA protein (Fuller et al. 1984).

In the uvrB gene two putative DnaA protein binding sites are located in inverted orientation in a region with potential stem-loop structure that includes part of P3, the most 5′ of the three functional promoters identified in this gene (see above) (van den Berg et al. 1985). The possible interaction of DnaA protein with the uvrB gene represents another potential mechanism for coupling uvrB expression to DNA replication. Fuller et al. (1984) have suggested that the replication of oriC during initiation of DNA synthesis could generate sequences that may act as a sink for high- affinity binding of DnaA protein, resulting in the derepression of genes negatively regulated by this protein. The uvrB gene (and perhaps other genes that are regulated by dnaA) might encode products that play a role in replication initiation. Thus, in the case of UvrB protein, we have a possible example of a gene product involved in both nucleotide excision repair and DNA replication, the enhanced expression of which is independently regulated for both functions by using distinct operator/promoter sites. In cells containing damaged DNA the amount of available UvrB protein may limit replication initiation during excision repair or vice versa, depending on the relative affinity of UvrB protein for the excision repair and replication complexes.

Finally, it is interesting to consider another gene involved in DNA replication in E. coli, mutations in which can result in an abnormal excision-repair phenotype. The dnaB gene of E. coli encodes a protein of molecular weight ≈50×103 essential for replication of the E. coli chromosome (Kornberg, 1980). A strain designated CM1031 carries a temperature-sensitive mutation in the dnaB gene (Bridges, 1975). At permissive temperatures, this strain has normal resistance to u.v. radiation but is deficient in host-cell reactivation of bacteriophage λ. Bridges et al. (1976) observed that this mutant loses sites sensitive to a dimer-specific enzyme probe at a reduced rate relative to the parental strain, suggesting a partial defect in excision repair. The possible involvement of DnaB protein in nucleotide excision repair represents a third potential component of the E. coli replication complex, which may be limited in cells exposed to DNA damage (Fig. 2).

Fig. 2.

The UvrD and UvrB proteins are known to be involved in nucleotide excision repair in E. coli, and may also be involved in DNA replication. DnaB protein is required for DNA replication, and may also be involved in nucleotide excision repair.

Fig. 2.

The UvrD and UvrB proteins are known to be involved in nucleotide excision repair in E. coli, and may also be involved in DNA replication. DnaB protein is required for DNA replication, and may also be involved in nucleotide excision repair.

The UvrA, UvrB and UvrC proteins

Isolation of the uvrA, uvrB and uvrC genes and their overexpression in E. coli has greatly facilitated the purification of the proteins they encode and their use in biochemical studies. The measured molecular weights of the UvrA, UvrB and UvrC proteins are ≈ 114× 103, ≈84 × 103 and ≈67× 103, respectively (Thomas et al. 1985), and agree well with values calculated from the coding regions of the cloned genes (103749, 76 118 and 66038) (Sancar et al. 1984; A. Sancar, personal communication).

Purified UvrA protein is a DNA-independent ATPase (Seeberg & Steinum, 1982). Neither of the other two Uvr proteins has demonstrable catalytic activity of any kind. However, as indicated above, a mixture of UvrA and UvrB proteins shows more ATPase activity than UvrA protein alone (Thomases al. 1985). It is not known whether this reflects stimulation of the activity associated with UvrA protein or the unmasking of an activity in the UvrB protein that is dependent on binding to UvrA protein. None of the Uvr proteins in isolation or in any pairwise combinations catalyses the incision of damaged DNA. But when u.v.-irradiated DNA is incubated with a mixture of all three Uvr proteins incision is observed and is strictly dependent on the presence of Mg2+ and ATP (Seeberg, 1978; Sancar & Rupp, 1983; Yeung et al. 1983). On the basis of the long-standing preconception of the molecular mechanism of nucleotide excision repair in E. coli discussed in the Introduction, it was anticipated that this reaction would generate incisions exclusively 5′ to sites of base damage, thus setting the stage in vivo for coordinated excision/resynthesis mediated by DNA polymerase I. Surprisingly, incision occurs on both sides of each lesion of a given DNA strand (Sancar & Rupp, 1983; Yeung et al. 1983). With respect to cyclobutyl pyrimidine dimers as well as so-called (6-4) or Py-C products (the two major photoproducts in DNA) the UvrABC catalytic activity hydrolyses the eighth phosphodiester bond 5′ and the fourth or fifth phosphodiester bond 3′ to the lesions (Sancar & Rupp, 1983). A fundamentally similar mode of incision in vitro has been observed with DNA containing monoadduct base damage caused by photoactivated psoralen or N-acetoxy-acetylaminofluorene (Sancaret al. 1985). The only difference is that with these substrates incisions 3′ to sites of base damage are exclusively at the fifth phosphodiester bond. The pattern of DNA incision is insensitive to the ratio of psoralen monoadducts and psoralen crosslinks and hence it has been suggested that DNA containing interstrand crosslinks is cut in the same way as DNA containing monoadduct or dipyrimidine base damage (Sancar et al. 1985). Incision of DNA by the purified UvrABC activity has been observed with a variety of other chemicals that produce bulky base adducts in DNA (Fuchs & Seeberg, 1984; Kacinski & Rupp, 1984; Husain et al. 1985) and, while not specifically demonstrated, it is a reasonable assumption that in all cases a bilateral incision mechanism operates.

The demonstration in vitro of bilateral DNA incision that is strictly dependent on all three Uvr proteins raises a number of interesting questions concerning its mechanism. How do the Uvr proteins interact with DNA and with one another? How is a catalytically active endonuclease constituted? How is incision apparently confined to the DNA strand containing sites of base damage? How are the bilateral incisions placed at precise locations with respect to sites of base damage? How does bilateral incision facilitate excision of base damage? Answers to these questions are beginning to emerge. There are no indications that the Uvr proteins constitute a preformed complex prior to incision of damaged DNA. On the contrary, the available evidence suggests that individual proteins bind to damaged DNA in a sequential fashion and that a catalytically active UvrABC complex is generated on the substrate (Weiss & Grossman, 1986; Yeung et al. 1986). UvrA protein binds preferentially to double-stranded u.v.-irradiated DNA relative to unirradiated DNA, and this binding is stimulated in the presence of ATP and inhibited by ADP (Weiss & Grossman, 1986; Yeung et al. 1986). Binding of ATP to UvrA protein results in conformational alterations of the protein that may be important for the constitution of a catalytically active UvrABC complex (Weiss & Grossman, 1986). Thus, in the presence of ATP or non-hydrolysable ATP analogues such as ATP[yS] UvrA protein is more readily iodinated at tyrosine residues. Furthermore, in the absence of ATP UvrA protein forms dimers that exist in an equilibrium state with monomers, whereas in the presence of ATP[γS] the protein exists almost exclusively in the dimerized form. The combination of UvrA protein with UvrC protein does not increase its binding affinity for u.v.-irradiated DNA, and UvrB protein alone does not bind to u.v.-irradiated DNA. However, when UvrA and UvrB proteins are mixed both proteins bind to u.v.-irradiated DNA to form a DNA-protein complex with a half-life significantly longer than that of the UvrA-DNA complex (Weiss & Grossman, 1986; Yeung et al. 1986). Once UvrA and UvrB proteins bind to u.v.- irradiated DNA the addition of UvrC protein results in incision. Evidence suggests that UvrC protein binds directly to the UvrAB-DNA complex without first binding to DNA and that the formation of a stable UvrAB-DNA complex is the rate-limiting step in the incision reaction catalysed by the UvrABC endonuclease (Weiss & Grossman, 1986; Yeung et al. 1986).

Following incision of u.v.-irradiated DNA by the UvrABC endonuclease in vitro the DNA-protein complex persists, thus limiting the turnover of Uvr proteins. However, the addition of UvrD protein and DNA polymerase I alters the stability of this complex and also has a significant effect on the incision reaction itself (Caron et al. 1985; Husain et al. 1985; Kumura et al. 1985). When the amount of UvrC protein is rate-limiting for the UvrABC endonuclease-catalysed incision reaction the addition of UvrD protein stimulates the incision of u.v.-irradiated DNA, suggesting that this protein causes UvrC protein in the complex to turn over (Caron et al. 1985). However, the complete turnover of UvrA, UvrB and UvrC proteins requires the addition of both UvrD protein and DNA polymerase I (Husain et al. 1985). The role of DNA polymerase I is at least partially dependent on concurrent DNA polymerization (i.e. repair synthesis), since less stimulation of nicking of u.v.-irradiated DNA is observed in the absence of deoxyribonucleoside triphosphates (Husain et al. 1985). T4 DNA polymerase or the Klenow fragment of DNA polymerase I can replace intact E. coli DNA polymerase I; however, E. coli Rep protein cannot substitute for UvrD protein (Husain et al. 1985). The complete reaction involving the four Uvr proteins and DNA polymerase I is not associated with an increase in the initial rate of DNA incision (Husain et al. 1985). Thus, UvrD protein and DNA polymerase I apparently do not influence the kinetics of formation of the UvrABC complex on damaged DNA, or its intrinsic endonucleolytic activity.

The precise mechanism of the release (excision) of the oligonucleotide created by bilateral incision of u.v.-irradiated DNA is not yet established. Studies by Caron et al. (1985) have shown that under non-denaturing conditions the oligonucleotide fragment is not released in vitro and is dependent on the addition of both DNA helicase II and DNA polymerase I. On the other hand, Husain et al. (1985) observed some removal of cisplatin adducts from DNA in the absence of the latter two proteins, but, as is true for the incision reaction, the extent of damage excision was stimulated by their addition.

Collectively the data summarized above suggest the following model for the molecular mechanism of nucleotide excision repair in E. coli (Husain et al. 1985; Weiss & Grossman, 1986; Yeung et al. 1986). UvrA protein binds ATP and undergoes a conformational change that favours the formation of protein dimers. In this state UvrA protein has a high affinity for sites in duplex DNA with conformational alterations in secondary structure. UvrB protein binds specifically to UvrA-DNA complexes and these complexes are recognized by UvrC protein, the binding of which generates a catalytically active endonuclease that nicks the DNA at precise locations relative to DNA adducts. The events that determine strand specificity for DNA incision and the locations of these incisions relative to each lesion are uncertain at present. However, it is presumably not pure coincidence that the distance separating the two incisions flanking each lesion approximates one helical turn of S-DNA. It is also likely that the conformational distortion generated by bulky base adducts is asymmetrical with respect to the helical axis of DNA and this may facilitate preferential binding of UvrA protein to the damaged strand. The structure of the UvrABC complex subsequently assembled on the DNA may provide for contact of catalytic sites separated by a distance that is close to a single helical turn. Alternatively, the binding of the UvrABC complex to DNA may unwind the DNA helix, generating regions of local denaturation precisely 12 (and in the case of pyrimidine dimers sometimes 13) nucleotides long. The enzyme may then cleave the DNA strand to which it is preferentially bound at the boundaries of the denaturation bubble. Following DNA incision UvrD protein alone (or in combination with DNA polymerase I) promotes turnover of UvrC protein, which is freed to interact with other UvrAB-DNA complexes. A complex consisting of UvrA and UvrB proteins bound to a potential oligonucleotide 12 (or 13) nucleotides long remains relatively stable until both UvrD protein and DNA polymerase I (or DNA polymerase II or HI) effect its displacement from the DNA duplex with concurrent repair synthesis and DNA ligation.

This model is consistent with the phenotype of mutants defective in the uvr or polA genes. uvrA, uvrB and uvrC strains are completely defective in DNA incision. Mutants defective in the uvrD or polA genes should not manifest a defect in the initial rate of DNA incision. However, because these genes are required for turnover of UvrA, UvrB and UvrC proteins, one would expect mutations in these genes to reduce the extent of DNA incision relative to wild-type strains. As indicated above, a reduced efficiency of DNA incision in a uvrD mutant was demonstrated some years ago by Ben-Ishai & Sharon (1981), and both uvrD and polA mutants have been shown to be deficient in the loss of thymine-containing pyrimidine dimers (excision) from DNA during post-irradiation incubation in vivo (see Friedberg, 1985).

The model presented above predicts that DNA incision, damage excision, repair synthesis (and presumably DNA ligation) are highly coordinated events possibly effected by a multiprotein complex (repairosome) that acts in a strictly processive fashion (Weiss & Grossman, 1986). Assuming stoichiometric interactions of Uvr proteins and DNA polymerase, and in the absence of competing reactions such as DNA replication, the rate of nucleotide excision repair in E. coli is presumably limited by the amount of UvrA, UvrB and UvrC protein, all of which are present at levels of <100 molecules per cell (Husain et al. 1985). DNA polymerase I and UvrD protein are present in larger amounts and are not expected to be rate-limiting for nucleotide excision repair in the presence of semiconservative DNA synthesis. However, if, as suggested earlier, UvrB is also involved in DNA replication and DnaB protein is involved in excision repair, these proteins could limit the rate of replication during nucleotide excision repair.

At the present time, E. coli is the only organism for which any detailed information exists on the biochemistry of nucleotide excision repair. However, recent years have witnessed interesting developments in at least two eukaryotic systems that offer the potential for exploring the generality of this process as we know it in E. coli.

The yeast S. cerevisiae

Close to 100 yeast mutants (some of which may represent alleles of the same gene) have been isolated with the phenotype of abnormal sensitivity to killing by DNA- damaging agents (see Haynes & Kunz, 1981, for a review). Using u.v. radiation as a source of DNA damage the sensitivity of some mutants known to be defective in different single genes was compared with that of mutants defective in more than one of these genes. If mutations in both genes impart no more u.v. sensitivity than mutations in either gene alone the genes are said to be epistatic, and the most obvious interpretation of an epistatic interaction is that the two genes function in the same biochemical pathway (see Haynes & Kunz, 1981, for a review). Such experiments, when combined with the results of studies that directly measure parameters of nucleotide excision repair in u.v.-irradiated yeast cells (e.g. loss of thymine- containing pyrimidine dimers or introduction of strand breaks during post-u.v.- incubation) have identified 10 loci unique to the nucleotide excision repair (so-called RAD3) epistasis group. Two other mutant loci also behave epistatically with mutants in this group, but are also epistatic with mutant loci in other groups, suggesting an involvement in more than one type of cellular response to DNA damage. One of these was originally designated but has been renamed RAD24 (F. Eckhardt- Schump, W. Siede & J. C. Game, personal communication). The rad24-1 mutant is epistatic to other mutants in the RAD3 epistasis group with respect to u.v. radiation sensitivity and is epistatic to mutants in the RAD52 group with respect to sensitivity to X- or y-radiation (F. Eckhardt-Schump, W. Siede & J. C. Game, personal communication). Similarly, a strain mutated in the CDC8 gene that encodes thymidylate kinase is u.v.-sensitive and epistatic to a radl mutant (belonging to the RAD3 epistasis group) (Prakash et al. 1979). In addition, this mutant shows reduced u.v. mutability, and in this regard is epistatic to rad6 from the RAD6 epistasis group (Prakash et al. 1979). Thus, at least 12 genetic loci may be involved in nucleotide excision repair in yeast (Fig. 3).

Fig. 3.

Genes in the RAD3 epistasis group are thought to be involved in nucleotide excision repair in yeast. The involvement of the two genes marked in parenthesis has not been firmly established.

Fig. 3.

Genes in the RAD3 epistasis group are thought to be involved in nucleotide excision repair in yeast. The involvement of the two genes marked in parenthesis has not been firmly established.

Mutations in five of these loci (RADI, RAD2, RAD3, RAD4, RADIO) confer marked sensitivity to killing by u.v. radiation and a complete defect in phenotypes associated with nucleotide excision repair. Mutations in five others (RAD 7, RAD14, RAD16, RAD23, MMS19) result in less sensitivity to u.v. radiation and retain a significant residual capacity for repair, though at levels less than those in wild-type cells (see Friedberg, 1986; Friedberg et al. 1986). (The nucleotide excision repair capacity of the rad24 and cdc8 mutants has not been reported.) This segregation of gene functions into those absolutely required for nucleotide excision repair and those that apparently are not resembles that which distinguishes the uvrA, uvrB and uvrC genes from the uvrD avApolA genes of E. coli, and suggests the possibility that the Radi, Rad2, Rad3, Rad4 and RadlO proteins are involved in the incision of DNA while the second group of gene products are involved in post-incision events. This comparison is not intended to suggest that the Radi, Rad2, Rad3, Rad4 and RadlO proteins function as a specific homologue of the UvrABC complex of E. coli or that the Rad7, Radl4, Radió, Rad23 and Mmsl9 proteins are strictly analogous to E. coli UvrD protein and DNA polymerase I.

The RAD genes and Rad proteins

The RADI, RAD2, RAD3 and RADIO genes have been cloned by screening yeast genomic libraries for multicopy plasmids containing sequences that complement the u.v. sensitivity of appropriate mutants (see Friedberg, 1986; Friedberg et al. 1986). Such screening failed to reveal recombinant plasmids containing the RAD4 gene. Recent studies in my laboratory (R. Fleer, C. Nicolet, G. Pure & E. C. Friedberg, unpublished) have shown that RAD4 is located very close to a gene called SPT2, and that a recombinant plasmid previously isolated by Roeder et al. (1985) contains the entire RAD4 gene. However, in this plasmid, as well as in other plasmids propagated in E. coli, the gene is mutationally inactivated. Deletion of defined regions of the mutant plasmid-borne gene and repair of the resultant gaps by gene transfer from yeast genomic sequences reconstitutes circular plasmids with a functional RAD4 gene that complements the u.v. sensitivity of all rad4 mutants tested (R. Fleer, C. Nicolet, G. Pure & E. C. Friedberg, unpublished data). However, propagating such plasmids inE. coli again results in inactivation of the gene. These results suggest that Rad4 protein is toxic to E. coli and explain the failure to isolate the functional gene on a plasmid from a yeast library cloned in this bacterium. The confines of the RAD4 gene have been established by insertional mutagenesis at defined sites and by mapping the transcriptional start-sites, and indicate that the gene is ≈2·3×103bp in size, thus yielding a calculated molecular weight of ≈90×103 for Rad4 protein.

The nucleotide sequences and predicted amino acid sequences of the RAD1, RAD2, RAD3 and RAD10 genes and the proteins they encode have been determined (Yang & Friedberg, 1984; Naumovski et al. 1985; Reynolds et al. 1985a,b; Nicolet et al. 1985; Weiss & Friedberg, 1985; Madura & Prakash, 1986) and have revealed several points of interest. (1) The four genes can encode proteins of calculated molecular weight ≈110×103, ≈118×103, ≈89×103 and ≈24×103, respectively. Hence, if the products of all 10 genes known to be involved in nucleotide excision repair assemble as a single multiprotein complex at sites of base damage in yeast chromatin, the size of such a complex is at least 500× 103Mr and could be as large as 106Mr. (2) Intervening sequences have not been detected in any of these genes and all are present in the yeast genome in single copy. (3) Hydropathicity profiles of the predicted polypeptides show a balanced distribution of hydrophobic and hydrophilic domains in the Radi, Rad3 and Rad 10 polypeptides. Rad2 protein appears to be unusually hydrophilic (Fig. 4). (4) We have not detected extensive nucleotide sequence homology between any of these genes, or with other genes evaluated by computer search. Furthermore, using the cloned RAD genes as probes we have not observed hybridization to total human, rodent, Drosophila or E. coli DNA. Several limited regions of amino acid sequence homology have been noted between the Rad proteins themselves and between one or more Rad proteins and other proteins. The most interesting of these are the following: (a) the tripeptide GKS (T or G) observed in the amino acid sequence of the E. coli Uvr proteins and DNA polymerase I (see Structural features, p. 4) is also common to the Rad3 (Reynolds et al. 1985a; Naumovski & Friedberg, 1986), Radi and RadlO proteins of.S. cerevisiae (Fig. 1), suggesting a convergent evolutionary relationship between these three yeast proteins and the excision repair proteins of E. coli. The homology between the yeast Rad3 protein and the E. coli UvrA, UvrB and UvrD proteins is particularly impressive. Over a stretch of 22 amino acids, 19 are common to two or more of these four proteins and the pentapeptide GS(T)GKT(S) is common to all of them. This has focused attention on the possibility that Rad3 protein may possess ATPase, GTPase and/or nucleotide binding activity (see below), (b) There is significant homology between the carboxy-terminal half of the Rad10 polypeptide and a region of a protein designated Erccl, which is believed to be involved in nucleotide excision repair of DNA in human cells (van Duin et al. 1986) (see below). Over a stretch of ≈100 consecutive amino acids, 39 are common to both proteins and a further 37 are closely related (van Duin et al. 1986). (c) A number of prokaryotic specific DNA binding proteins share a consensus amino acid sequence consistent with the α-helix-turn-α- helix secondary structure that is characteristic of their DNA binding domain. Among the lower eukaryotes many of the proteins with so-called horneo boxes match this consensus rather well, as do regions in the yeast DNA binding proteins Matα1 and Matα2. Comparison of a number of these proteins with Rad10 and Rad3 suggests that these two proteins may interact with DNA through a α-helix-turn-α-helix motif (van Duin et al. 1986; Naumovski & Friedberg, 1986).

Fig. 4.

Hydropathicity profiles of the Radi, Rad2, Rad3 and RadlO proteins. The light areas highlight peaks with values greater than +2·5 or −2·5.

Fig. 4.

Hydropathicity profiles of the Radi, Rad2, Rad3 and RadlO proteins. The light areas highlight peaks with values greater than +2·5 or −2·5.

In summary, these amino acid sequence comparisons suggest that the E. coli and some of the yeast nucleotide excision repair proteins share a common domain, which may be important functionally in nucleotide binding and/or nucleotide triphosphatase activity. In addition, Rad3 and Rad10 proteins may bind to DNA, perhaps in a sequence-specific fashion.

The functional relationships suggested by these homologies can be explored in a number of ways. One is to determine whether any of the Rad proteins can complement phenotypes related to excision repair defects in E. coli and mammalian cells. To the extent that such experiments have been completed no interspecies complementation has been reported. The RAD3 and RAD10 genes have been expressed in E. coli and yield proteins of the expected size, yet these proteins do not complement the u.v. sensitivity of uvrA, uvrB, uvrC or uvrD mutants (L. Naumovski, W. A. Weiss & E. C. Friedberg, unpublished observations). Similarly, the uvrA and uvrB genes have been expressed in rad1, rad2, rad3 and rad10 strains without detectable phenotypic complementation of these mutants (Planque et al. 1984). The RAD3 and RAD10 genes have also been tailored into mammalian cell expression vectors (L. Couto, G. Chu, R. Schultz, P. Berg & E. C. Friedberg, unpublished observations). At the time of writing, it has not been definitively established that the respective Rad proteins are actually expressed in mammalian cells transfected with these recombinant plasmids. Nonetheless, no enhancement of the u.v. resistance of xeroderma pigmentosum (XP) group A cells (by RAD3) or of a Chinese hamster ovary (CHO) cell line designated UV20 (see below) (by RADIO) has been observed.

A second way of exploring structural relationships between proteins from different organisms is by using specific antibodies. We have recently raised rabbit antisera that react specifically with yeast Rad2 and Rad3 proteins (L. Naumovski, C. Nicolet & E. C. Friedberg, unpublished observations). The antiserum containing rabbit anti- Rad3 antibodies does not react with purified UvrA, UvrB or UvrC proteins by immunoblotting (C. Backendorf, L. Naumovski & E. C. Friedberg, unpublished observations). Reactions of both antisera against extracts of mammalian cells and of anti-Rad2 antiserum against Uvr proteins, are in progress.

Finally, it is important to determine whether any of the Rad proteins catalyse reactions suggested by sequence homology with other proteins. Rad3 protein has been extensively purified, but no ATPase or GTPase activity has been detected (L. Naumovski & E. C. Friedberg, unpublished observations). However, we have not yet excluded the possibility that such activity is dependent on the association of Rad3 with other Rad protein(s), or on some other specific mechanism of activation. Studies on nucleotide and DNA binding by Rad3 protein and on DNA helicase activity in this protein are in progress.

RAD3 is an essential gene in S. cerevisiae

Disruption (as opposed to point mutagenesis) of the RAD3 gene is lethal to haploid cells, indicating that this gene is essential for viability. None of the other RAD genes under study has this property. The nature of the essential function is not known. However, the observation that rad3 alleles carrying point mutations are viable, but defective in nucleotide excision repair, suggests that Rad3 protein may have two distinct activities. In an effort to gain insight into the nature of these activities and their localization at the level of the primary structure of Rad3 protein, we mutagenized a number of sites in the cloned RAD3 gene and examined their effect on the phenotypes of u.v. sensitivity and viability (Naumovski & Friedberg, 1986). Single point mutations in the putative nucleotide or DNA binding domains had no effect on the essential function of Rad3; indeed, the essential function of Rad3 protein is very resistant to inactivation by single point mutations and the only such mutant isolated to date contains a temperature-sensitive point mutation at codon position 73. A site of tandem point mutations affecting two adjacent codons in the putative DNA binding domain also inactivates the essential function, but the site specificity of these mutations is uncertain, pending study of the effect of tandem mutations in other regions of the protein. The excision repair function of Rad3 protein is inactivated by single point mutations at many sites (including both the putative nucleotide binding and DNA binding domains), an observation that explains the facile isolation of viable, excision repair-defective mutants. Collectively, these results suggest that whatever the nature of the nucleotide excision repair function of the Rad3 protein, it is highly sensitive to conformational changes, whereas the essential function of the protein is not. Hence, it is likely that the type of protein-protein and/or protein-DNA interactions that characterize the two Rad3 functions are different.

The essentiality of the RAD3 gene may constitute an example in yeast of the suggestion made earlier with respect to E. coli, i.e. that some proteins involved in nucleotide excision repair are also involved in other DNA transactions such as replication. As indicated above, we have isolated a temperature-sensitive rad3 mutant that is defective for growth at non-permissive temperatures. Studies on DNA replication in this mutant at permissive and restrictive temperatures may be illuminating.

The RAD2 gene is inducible by DNA damage

As indicated earlier, the E. coli UvrA, UvrB and UvrC proteins are constitutively expressed in very small amounts (≈10−20 copies per cell). In the case of the first two proteins this level is amplified ≈fivefold in cells exposed to DNA damage or other treatments that interfere with normal semiconservative DNA synthesis. At least some of the yeast RAD genes are also weakly expressed. Accurate quantitative measurements of transcriptional activity are difficult to obtain; however, qualitative observations from DNA-RNA hybridization experiments indicate the presence of very small amounts of RAD1, RAD2, RAD3 and RAD10 transcripts (L. Naumovski, C. Nicolet, E. Yang & E. C. Friedberg, unpublished observations). In addition, fusions of the promoter and 5′ coding region of these genes to the E. coli lacZ coding region results in very limited expression of E. coli β-galactosidase relative to fusions with highly expressed yeast genes (Robinson et al. 1986).

When yeast cells containing a single integrated copy of a RAD2-lacZ fusion plasmid (or an autonomously replicating multicopy fusion plasmid) are exposed to treatments known to induce E. coli SOS genes, fivefold enhanced expression of β-galactosidase is observed (Robinson et al. 1986). Cells transformed with RAD1-, RAD3- or RAD10-lacZ fusions do not show this result. Consistent with the notion of enhanced gene expression, untransformed cells exposed to 4-nitroquinoline-l-oxide (4NQO) show increased levels of RAD2 mRNA. The levels of RADIO messenger are unaffected by treatment of cells with 4NQO, while RAD3 and RAD4 transcripts actually decrease in amount (Robinson et al. 1986).

The regulation of this inducible response to DNA damage is currently under investigation. Investigators have shown that other yeast genes are induced by DNA damage. These include the CDC9 gene, which encodes DNA ligase (Barker et al. 1985; Peterson et al. 1985) the RAD51 and RAD54 genes (D. Schild & R. K. Mortimer, personal communication), which are involved in genetic recombination and which confer sensitivity to ionizing radiation when mutated, and a group of unidentified genes referred to as damage-inducible (DIN) (Ruby & Szostak, 1985) or DNA damage-responsive (DDR) genes (McClanahan & McEntee, 1984, 1986). Whether or not some or all of these genes belong to the RAD2 regulon remains to be determined.

Nucleotide excision repair in mammalian cells

Information on the molecular biology of nucleotide excision repair in mammalian cells is very limited. Genetic analyses in human and in rodent cells support the notion of biochemical complexity evident from studies in yeast. Somatic cell hybridization using fibroblasts from patients with the human disease xeroderma pigmentosum (XP) has revealed nine complementation groups, suggesting that at least nine distinct genes are involved in nucleotide excision repair in humans (see Friedberg, 1985). Similar analyses with u.v.-sensitive and excision repair-defective CHO cells has demonstrated five complementation groups to date (Thompson et al. 1985<z). Systematic hybridization between human and CHO cells representative of all these complementation groups has not been completed. However, the hybrids tested to date show enhanced levels of u.v. resistance (Thompson et al. 1985<z), suggesting that the human and CHO mutations examined are in different loci. It seems improbable to me that the molecular mechanism of nucleotide excision repair in these two mammals is completely distinct. Hence, the observation that related human and hamster genes have not been identified raises the interesting and awesome possibility that the spectrum of excision repair genes is larger than that revealed by either of the existing complementation groups alone. Since a case has been made for the involvement of at least 12 genes in nucleotide excision repair in yeast, the operational definition of 14 nucleotide excision repair genes in mammalian cells (9 by analysis of XP cells and 5 by analysis of CHO cells) is perhaps not unreasonable. In this regard, it is important to bear in mind that the CHO mutants were selected in the laboratory by screening mutagenized immortalized cells for enhanced u.v. sensitivity, whereas the XP cell lines are derived from human diploid strains established from biopsy of human patients. The human cell lines may therefore reflect a bias against mutations incompatible with normal human embryogenesis and development, while the CHO lines may (for reasons that are not clear) represent a bias against mutations in genes that in human cells characterize the disease XP.

Human excision repair genes and proteins

Attempts to isolate human genes by phenotypic complementation of XP cells in culture have not been successful (see Schultz et al. 1985). On the other hand, complementation of u.v.-sensitive CHO mutants by transfection with human DNA has been observed in several studies (Rubin et al. 1983, 1985; Westerveldet al. 1984) and has facilitated the isolation of a DNA fragment containing a gene called ERCC-1 (excision repair complementing defective repair in Chinese hamster cells). Transfection of CHO cells from complementation group 2 with cosmids containing this gene results in enhanced resistance to killing by mitomycin C or u.v. radiation, as well as enhanced levels of unscheduled DNA synthesis and excision of thymine- containing pyrimidine dimers (Westerveld et al. 1984; Rubin et al. 1985).

Using a portion of the ERCC-1 gene as a hybridization probe, van Duin and his colleagues (1986) isolated a series of overlapping cDNAs from which a complete cDNA was reconstructed. This cDNA complements the u.v. sensitivity of appropriate CHO mutants, but to date has not been shown to complement human XP cells (D. Bootsma, personal communication). DNA sequence analysis of the ERCC-1 cDNA revealed an open reading frame consisting of 297 codons, which could encode a protein of calculated molecular weight 32562. As indicated previously, comparison of the predicted amino acid sequence of the Erccl polypeptide reveals considerable homology with the yeast RadlO protein. It is also remarkable that a cDNA missing 302 bp from the 5′ end of the gene (including 162 bp of 5′ coding sequence) still complements the phenotype of mutant CHO cells (van Duin et al. 1986). At the time of writing, it has not been established whether the ERCC-1 gene complements the phenotype of yeast radlO mutants. Attempts to complement CHO cells from complementation group 2 with the cloned yeast RADIO gene are in progress in my laboratory. The human ERCC-1 gene has been mapped to chromosome 19, consistent with a recent report that this chromosome complements the phenotype of a CHO mutant from complementation group 2 (Thompson et al. 19856).

The use of CHO (and possibly other rodent) cells, in which stable transformation by DNA-mediated transfection occurs at a reasonable frequency, would appear to be a useful procedure for screening the human genome for complementing sequences. One would expect that human genes identified in this way are involved in nucleotide excision repair in normal human cells, and the amino acid sequence homology between the human Ercc-1 and yeast RadlO proteins is certainly consistent with this expectation. However, in view of the failure to demonstrate overlapping mutations in human XP and CHO cells, it would not be surprising if such human sequences do not complement XP cells and hence turn out to be uninformative with respect to the molecular basis of this human disease. Thus, there are compelling imperatives for the cloning of human genes by direct complementation of the phenotype of XP cells. In this regard, new strategies for phenotypic complementation by DNA transfer may be required, since to date XP cells have proven particularly resistant to conventional approaches to phenotypic complementation.

The author acknowledges the contributions of numerous post-doctoral fellows and students whose work on nucleotide excision repair in yeast and mammalian cells is described here. I also thank Aziz Sancar, Claude Backendorf and Lawrence Grossman for unpublished information, Jane Cooper, Linda Couto, Reinhard Fleer, Charles Nicolet, Gordon Robinson, Roger Shultz and William Weiss for critical review, Priscilla Cypiot for generating the hydropathicity profiles shown in Fig. 4, and Jean Oberlindacher and Margaret Beers for preparation of the manuscript. Work from the author’s laboratory is supported by research grants from the National Cancer Institute, USPHS, the Council for Tobacco Research and the American Cancer Society, and by contract with the US Department of Energy.

Arikan
,
E.
,
Kulkarni
,
M. S.
,
Thomas
,
D. C.
&
Sancar
,
A.
(
1986
).
Sequences of the E. coli uvrB gene and protein
.
Nucl. Acids Res.
14
,
2637
2650
.
Arthur
,
H. M.
,
Bramhill
,
D.
,
Eastlake
,
P. B.
&
Emmerson
,
P. T.
(
1982
).
Cloning of the uvrDgene of E. coli and identification of the product
.
Gene
19
,
285
295
.
Backendorf
,
C.
,
Spaink
,
H.
,
Barbeiro
,
A. P.
&
van de Putte
,
P.
(
1986
).
Structure of the uvrBgene of Escherichia coli. Homology with other DNA repair enzymes and characterization of the uvrB5 mutation
.
Nucl. Acids Res.
14
,
2877
2890
.
Barker
,
D. G.
,
White
,
J. H. M.
&
Johnston
,
L. H.
(
1985
).
The nucleotide sequence of the DNA ligase gene (CDC9) from Saccharomyces cerevisiae: a gene which is cell-cycle regulated and induced in response to DNA damage
.
Nucl. Acids Res.
13
,
8323
8337
.
Ben-Ishai
,
R.
&
Sharon
,
R.
(
1981
). On the nature of the repair deficiency in E. coli uvrE. In
Chromosome Damage and Repair
(ed. E. Seeberg &
K.
Kleppe
), pp.
147
151
.
New York
:
Plenum Press
.
Bridges
,
B. A.
(
1975
).
An ultraviolet-resistant tsDNA mutant of Escherichia coli deficient in host cell reactivation ability for bacteriophage lambda and showing hypersensitivity towards induction of Weigle-reactivation
.
Mutat. Res.
29
,
489
492
.
Bridges
,
B. A.
,
Mottershead
,
R. P.
&
Lehmann
,
A. R.
(
1976
).
Error-prone DNA repair in Escherichia coli. IV. Excision repair and radiation-induced mutation in a dnaB strain
.
Biol. Zbl.
95
,
393
403
.
Caron
,
P. R.
,
Kushner
,
S. R.
&
Grossman
,
L.
(
1985
).
Involvement of helicase II (uvrD gene product) and DNA polymerase I in excision mediated by the uvrABC protein complex
.
Proc, natn. Acad. Sci. U.S A..
82
,
4925
4929
.
Cleaver
,
J. E.
(
1986
).
Xeroderma pigmentosum
. In
Photomedicine
(ed. E. Ben-Hurand & I. Rosenthal),
Boca
Raton
, Fla: CRC Press (
in
press
).
Easton
,
A. M.
&
Kushner
,
S. R.
(
1983
).
Transcription of the uvrD gene of Escherichia coli is controlled by the lexA repressor and by attenuation
.
Nucl. Acids Res.
11
,
8627
8641
.
Finch
,
P. W.
&
Emmerson
,
P. T.
(
1984
).
The nucleotide sequence of the uvrD gene of E. coli. Nucl. Acids Res.
12
,
5789
5799
.
Forster
,
J. W.
&
Strike
,
P.
(
1985
).
Organization and control of the Escherichia coli uvrC gene
.
Gene
35
,
71
82
.
Friedberg
,
E. C.
(
1985
).
DNA Repair.
New York
:
W. H. Freeman and Co
.
Friedberg
,
E. C.
(
1986
).
Nucleotide excision repair of DNA in eukaryotes: comparisons between human cells and yeast
.
Cancer Surveys
4
,
529
555
.
Friedberg
,
E. C.
,
Fleer
,
R.
,
Naumovski
,
L.
,
Nicolet
,
C.
,
Robinson
,
G. W.
,
Weiss
,
W. A.
&
Yang
,
E.
(
1986
). Nucleotide excision repair genes from the yeast Saccharomyces cerevisiae. In
Mechanisms of Antimutagenesis and Anticarcinogenesis
(ed.
D. M.
Shankel
,
P.
Hartman
, T. Kada &
A.
Hollaender
), pp.
231
242
.
New York
:
Plenum Press
.
Fuchs
,
R. P. P.
&
Seeberg
,
E.
(
1984
).
pBR322 plasmid DNA modified with 2-acetyl- aminofluorene derivatives: transforming activity and in vitro strand cleavage by the Escherichia coli uvrABC endonuclease
.
EMBOJ.
3
,
757
760
.
Fuller
,
R. S.
,
Funnell
,
B. E.
&
Kornberg
,
A.
(
1984
).
The dnaA protein complex with the E. coli chromosomal replication origin (oriC) and other DNA sites
.
Cell
38
,
889
900
.
Haynes
,
R. H.
&
Kunz
,
B. A.
(
1981
).
DNA repair and mutagenesis in yeast
. In
The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance
(ed.
J. N.
Strathern
, E. W. Jones &
J. R.
Broach
), pp.
371
-
414
. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Horiuchi
,
T.
&
Nagata
,
T.
(
1973
).
Mutations affecting growth of the Escherichia coli cell under a condition of DNA polymerase I deficiency
.
Molec. gen. Genet.
123
,
89
—110.
Husain
,
L
,
Chaney
,
S. G.
&
Sancar
,
A.
(
1985
).
Repair of cis-platinum-DNA adducts by ABC exinuclease in vivo and in vitro.
J. Bact.
163
,
817
—823.
Husain
,
L
,
Van Houten
,
B.
,
Thomas
,
D. C.
,
Abdel-Monem
,
M.
&
Sàncar
,
A.
(
1985
).
Effect of DNA polymerase I and DNA helicase II on the turnover rate of UvrABC excision nuclease
.
Proc. natn. Acad. Sci. U.S A.
82
,
6774
6778
.
Husain
,
L
,
Van Houten
,
B.
,
Thomas
,
D. C.
&
Sancar
,
A.
(
1986
).
Sequences of E. coli uvrAgene and protein reveal two potential ATP binding sites. JC biol. Chem.
261
,
4895
4901
.
Joyce
,
C. M.
,
Kelley
,
W. S.
&
Grindley
,
N. D. F.
(
1982
).
Nucleotide sequence of the Escherichia coli polA gene and primary structure of DNA polymerase I
.
J. biol. Chem
.
251
,
1958
-
1964
.
Kacinski
,
B. M.
&
Rupp
,
W. D.
(
1984
).
Interaction of the UVRABC endonuclease in vivo and in vitro with DNA damage produced by antineoplastic anthracyclines
.
Cancer Res.
44
,
3489
3492
.
Kenyon
,
C. J.
&
Walker
,
G. C.
(
1980
).
DNA-damaging agents stimulate gene expression at specific loci in Escherichia coli.
Proc. natn. Acad. Sci. U.S.A.
77
,
2819
2823
.
Klinkert
,
M.-Q.
,
Klein
,
A.
&
Abdel-Monem
,
M.
(
1980
).
Studies on the functions of DNA helicase I and DNA helicase II of Escherichia coli.
J. biol. Chem.
255
,
9746
9752
.
Kornberg
,
A.
(
1980
).
DNA Replication. New York: W. H. Freeman
.
Kuhn
,
B.
&
Abdel-Monem
,
M.
(
1982
).
DNA synthesis at a fork in the presence of DNA helices
.
Eur.J. Biochem.
125
,
63
68
.
Kumura
,
K.
&
Sekiguchi
,
M.
(
1984
).
Identification of the uvrD gene product of Escherichia colias DNA helicase II and its induction by DNA-damaging agents
.
J. biol. Chem.
259
,
1560
1565
.
Kumura
,
K.
,
Sekiguchi
,
M.
, Steinum, A.-L. &
Seeberg
,
E.
(
1985
).
Stimulation of the UvrABC enzyme-catalyzed repair reactions by the UvrD protein (DNA helicase II). Nucl. Acids Res.
13
,
1483
1492
.
Kyte
,
J.
&
Doolittle
,
R. F.
(
1982
).
A simple method for displaying the hydropathic character of a protein
.
J. molec. Biol.
157
,
105
132
.
Lu
,
A.-L.
,
Clark
,
S.
&
Modrich
,
P.
(
1983
).
Methyl-directed repair of DNA base-pair mismatches in vitro.
Proc. natn. Acad. Sci. U.S A.
80
,
4639
4643
.
Madura
,
K.
&
Prakash
,
S.
(
1986
).
Nucleotide sequence, transcript mapping, and regulation of the RAD2 gene of Saccharomyces cerevisiae.
J. Bad.
166
,
914
923
.
Maples
,
V. F.
&
Kushner
,
S. R.
(
1982
).
DNA repair in Escherichia coli’. Identification of the uvrD gene product
.
Proc. natn. Acad. Sci. U.S A.
79
,
5616
5620
.
McClanahan
,
T.
&
McEntee
,
K.
(
1984
).
Specific transcripts are elevated in Saccharomyces cerevisiae in response to DNA damage
.
Molec. cell. Biol.
4
,
2356
2363
.
McClanahan
,
T.
&
McEntee
,
K.
(
1986
).
DNA damage and heat shock dually regulate genes in Saccharomyces cerevisiae.
Molec. cell. Biol.
6
,
90
96
.
Morimyo
,
M.
&
Shimazu
,
Y.
(
1976
).
Evidence that the gene uvrB is indispensable for a polymerase I deficient strain of Escherichia coli K-12
.
Mol. gen. Genet.
147
,
243
250
.
Naumovski
,
L.
,
Chu
,
G.
,
Berg
,
P.
&
Friedberg
,
E. C.
(
1985
).
RAD3 gene of Saccharomyces cerevisiae: nucleotide sequence of wild-type and mutant alleles, transcript mapping and aspects of gene regulation
.
Molec. cell. Biol.
5
,
17
26
.
Naumovski
,
L.
&
Friedberg
,
E. C.
(
1986
).
Analysis of the essential and excision repair functions of the RAD3 gene of S. cerevisiae by mutagenesis
.
Molec. cell. Biol.
6
,
1218
1227
.
Nicolet
,
C. M.
,
Chenevert
,
J. M.
&
Friedberg
,
E. C.
(
1985
).
The RAD2 gene of Saccharomyces cerevisiae: nucleotide sequence and transcript mapping
.
Gene
36
,
225
234
.
Peterson
,
T. A.
,
Prakash
,
L.
, Prakash., S.,
Osley
,
M. A.
&
Reed
,
S. I.
(
1985
).
Regulation of CDC9, the Saccharomyces cerevisiae gene that encodes DNA ligase
.
Molec. cell. Biol.
5
,
226
235
.
Planque
,
K.
,
Backendorf
,
C. F.
,
Lekkerkerk
,
J.
&
van de Putte
,
P.
(
1984
).
Expression of bacterial uvrA and uvrB in Saccharomyces cerevisiae.
Twelfth Int. Conf. Yeast Genet. Mol. Biol. Edinburgh, p. 211 (Abstr
.).
Prakash
,
L.
,
Hinkle
,
D.
&
Prakash
,
S.
(
1979
).
Decreased UV mutagenesis in cdc-8, a DNA replication mutant of Saccharomyces cerevisiae.
Molec. gen. Genet.
172
,
249
258
.
Reynolds
,
P.
,
Higgins
,
D. R.
,
Prakash
,
L.
&
Prakash
,
S.
(
1985a
).
The nucleotide sequence of the RAD3 gene of Saccharomyces cerevisiae: a potential adenine nucleotide binding amino acid sequence and a non-essential acidic carboxyl terminal region
.
Nucl. Acids Res.
13
,
2357
2372
.
Reynolds
,
P.
,
Prakash
,
L.
,
Dumais
,
D.
,
Perozzi
,
G.
&
Prakash
,
S.
(
1985b
).
Nucleotide sequence of the RADIO gene of Saccharomyces cerevisiae.
EMBOJ.
4
,
3549
3552
.
Robinson
,
G. W.
,
Nicolet
,
C. M.
,
Kalainov
,
D.
&
Friedberg
,
E. C.
(
1986
).
A yeast excision repair gene is inducible by DNA damaging agents
.
Proc. natn. Acad. Sci. U.S A.
83
,
1842
1846
.
Roeder
,
G. S.
,
Beard
,
C.
,
Smith
,
M.
&
Keranen
,
S.
(
1985
).
Isolation and characterization of the SPT2 gene, a negative regulator of Ty-controlled yeast gene expression
.
Molec. cell. Biol.
5
,
1543
1553
.
Rubin
,
J. S.
,
Joyner
,
A. L.
,
Bernstein
,
A.
&
Whitmore
,
G. F.
(
1983
).
Molecular identification of a human DNA repair gene following DNA-mediated gene transfer
.
Nature, Lond.
306
,
206
208
.
Rubin
,
J. S.
,
Prideaux
,
V. R.
,
Willard
,
H. F.
,
Dulhanty
,
A. M.
,
Whitmore
,
G. F.
&
Bernstein
,
A.
(
1985
).
Molecular cloning and chromosomal localization of DNA sequences associated with a human DNA repair gene
.
Molec. cell. Biol.
5
,
398
405
.
Ruby
,
S.
&
Szostak
,
J. W.
(
1985
).
Specific Saccharomyces cerevisiae genes are expressed in response to DNA-damaging agents
.
Molec. cell. Biol.
5
,
75
84
.
Sancar
,
A.
,
Clarke
,
N. D.
,
Griswold
,
J.
,
Kennedy
,
W. J.
&
Rupp
,
W. D.
(
1981b
).
Identification of the uvrB gene product
.
J. molec. Biol.
148
,
63
76
.
Sancar
,
A.
,
Franklin
,
K. A.
,
Sancar
,
G.
&
Tang
,
M.-S.
(
1985
).
Repair of psoralen and acetylaminofluorene DNA adducts by ABC excinuclease
.
J. molec. Biol.
184
,
725
734
.
Sancar
,
A.
&
Rupp
,
W. D.
(
1983
).
A novel repair enzyme: UVRABC excision nuclease of Escherichia coli cuts a DNA strand on both sides of the damaged region
.
Cell
33
,
249
260
.
Sancar
,
A.
,
Sancar
,
G. B.
,
Little
,
J. W.
&
Rupp
,
W. D.
(
1982zz
).
The uvrB gene of Escherichia coli has both fexA-repressed and fexA-independent promoters
.
Cell
28
,
523
530
.
Sancar
,
A.
,
Sancar
,
G. B.
,
Rupp
,
W. D.
,
Little
,
J. W.
&
Mount
,
D. W.
(
1982b
).
LexA protein inhibits transcription of the E. coli uvrA gene in vitro.
Nature, Lond.
298
,
96
98
.
Sancar
,
A.
,
Wharton
,
R. P.
,
Seltzer
,
S.
,
Kacinski
,
B. M.
,
Clarke
,
N. D.
&
Rupp
,
W. D.
(
1981a
).
Identification of the uvrA gene product
.
J. molec. Biol.
148
,
45
62
.
Sancar
,
G. B.
,
Sancar
,
A.
&
Rupp
,
W. D.
(
1984
).
Sequences of the E, coli uvrC gene and protein
.
Nucl. Acids Res.
12
,
4593
4608
.
Schultz
,
R. A.
,
Barbis
,
D.
&
Friedberg
,
E. C.
(
1985
).
Studies on gene transfer and on reversion to UV resistance in xeroderma pigmentosum cells
.
Somat. cell, molec. Genet.
11
,
617
624
.
Sedgwick
,
S. G.
(
1986
). Stability and change through DNA repair. In
Accuracy in Molecular Processes
(ed.
D. J.
Galas
, T. B. L. Kirkwood &
R. F.
Rosenberger
).
London
:
Chapman and Hall (in press
).
SEEBERG
,
E.
(
1978
).
Reconstitution of an Escherichia coli repair endonuclease activity from the separated uvrA+ and uvrB+/uvrC+ gene products
.
Proc. natn. Acad. Sci. U.S A.
75
,
2569
2573
.
Seeberg
,
E.
(
1981
).
Multiprotein interactions in strand cleavage of DNA damaged by UV and chemicals
.
Prog. nucl. Acid Res. molec. Biol.
26
,
217
226
.
Seeberg
,
E.
,
Nissen-Meyer
,
J.
&
Strike
,
P.
(
1978
).
Incision of ultraviolet-irradiated DNA by extracts of E. coli requires three different gene products
.
Nature, Lond.
263
,
524
526
.
Seeberg
,
E.
&
Steinum
,
A.-L.
(
1982
).
Purification and properties of the uvrA protein from Escherichia coli.
Proc. natn. Acad. Sci. U.SA.
79
,
988
992
.
Shizuya
,
H.
&
Dykhuizen
,
D.
(
1972
).
Conditional lethality of deletions which include uvrB in strains of Escherichia coli lacking deoxyribonucleic acid polymerase I
.
J. Bad.
112
,
676
681
.
Siegel
,
E. C.
(
1973
).
Ultraviolet-sensitive mutator mutU4of Escherichia coli inviable with polA. J. Bad.
113
,
161
166
.
Siegel
,
E. C.
(
1983
).
The Escherichia coli uvrD gene is inducible by DNA damage
.
Molec. gen. Genet.
191
,
397
400
.
Smirnov
,
G. B.
,
Filkova
,
E. V.
,
Skavronskaya
,
A. G.
,
Saenko
,
A. S.
&
Sinzinis
,
B. I.
(
1973
).
Loss and restoration of viability of E. coli due to combinations of mutations affecting DNA polymerase I and repair activities
.
Molec. gen. Genet.
121
,
139
—150.
Thomas
,
D. C.
,
Levy
,
M.
&
Sancar
,
A.
(
1985
).
Amplification and purification of UvrA, UvrB, and UvrC proteins of Escherichia coli.
J. biol. Chem.
260
,
9875
9883
.
Thompson
,
L. H.
,
Mooney
,
C. L.
&
Brookman
,
K. W.
(
1985a
).
Genetic complementation between UV sensitive CHO mutants and xeroderma pigmentosum fibroblasts
.
Mutat. Res.
150
,
423
429
.
Thompson
,
L. H.
,
Mooney
,
C. L.
,
Burkhart-Schultz
,
K.
,
Carrano
,
A. V.
&
Siciliano
,
M. J.
(
1985b
).
Correction of a nücleotide-excision-repair mutation by human chromosome 19 in hamster-human hybrid cells
.
Somat. Cell molec. Genet.
11
,
87
92
.
van den Berg
,
E. A.
,
Geerse
,
R. H.
,
Memelink
,
J.
,
Bovenberg
,
R. A. L.
,
Magnee
,
F. A.
&
van de Putte
,
P.
(
1985
).
Analysis of regulatory sequences upstream of the E. coli uvrB gene; involvement of the DnaA protein
.
Nucl. Acids Res.
13
,
1829
1840
.
van den Berg
,
E. A.
,
Geerse
,
R. H.
,
Pannekoek
,
H.
&
van de Putte
,
P.
(
1983
).
In vivotranscription of the E. coli uvrB gene: both promoters are inducible by UV
.
Nucl. Acids Res.
11
,
4355
4363
.
van Duin
,
M.
,
de Wit
,
J.
,
Odijk
,
H.
,
Westerveld
,
A.
,
Yasui
,
A.
,
Koken
,
M.
,
Hoeiimakers
,
J. H. J.
&
Bootsma
,
D.
(
1986
).
Molecular characterization of the human excision repair gene ERCC-1: cDNA cloning and amino acid homology with the yeast DNA repair gene RADIO. Cell
44
,
913
923
.
van Sluis
,
C. A.
,
Moolenaar
,
G. F.
&
Backendorf
,
C.
(
1983
).
Regulation of the uvrC gene of Escherichia coli K12: localization and characterization of a damage-inducible promoter
.
EMBOJ.
12
,
2313
2318
.
Walker
,
G. C.
(
1985
).
Inducible DNA repair systems. A
.
Rev. Biochem.
54
,
425
454
.
Walker
,
G. C.
,
Marsh
,
L.
&
Dodson
,
L. A.
(
1985
).
Genetic analysis of DNA repair: inference and extrapolation. A
.
Rev. Genet.
19
,
103
126
.
Walker
,
J. E.
,
Saraste
,
M.
,
Runswick
,
M. J.
&
Gay
,
N. J.
(
1982
).
Distantly related sequences in the α- and β-subunits of ATP synthase, myosin, kinase and other ATP-requiring enzymes and a common nucleotide binding fold
.
EMBOJ.
1
,
945
951
.
Weiss
,
B.
&
Grossman
,
L.
(
1986
).
Phosphodiesterases involved in DNA repair
.
Adv. Enzymol.(in press)
.
Weiss
,
W. A.
&
Friedberg
,
E.
(
1985
).
Molecular cloning and characterization of the yeast RADIOgene and expression of RADIO protein in E. coli. EMBOJ.
4
,
1575
1582
.
Westerveld
,
A.
,
Hoeijmakers
,
J. H. J.
,
van Duin
,
M.
,
de Wit
,
J.
,
Odijk
,
H.
, Pastink,
A.
Wood
, R. D. &
Bootsma
,
D.
(
1984
).
Molecular cloning of a human DNA repair gene
.
Nature, Land.
310
,
425
428
.
Yang
,
E.
&
Friedberg
,
E. C.
(
1984
).
Molecular cloning and nucleotide sequence analysis of the Saccharomyces cerevisiae RADI gene
.
Molec. cell. Biol.
4
,
2161
2169
.
Yeung
,
A. T.
,
Mattes
,
W. B.
&
Grossman
,
L.
(
1986
).
Protein complexes formed during the incision reaction catalyzed by the Escherichia coli UvrABC endonuclease
.
Nucl. Acids Res.
14
,
2567
2582
.
Yeung
,
A. T.
,
Mattes
,
W. B.
,
Oh
,
E. Y.
&
Grossman
,
L.
(
1983
).
Enzymatic properties of purified Escherichia coli uvrABC proteins
.
Proc. natn. Acad. Sci. U.S A..
80
,
6157
6161
.
Yoakum
,
G. H.
&
Grossman
,
L.
(
1981
).
Identification of the E. coli UvrC protein
.
Nature, Land.
292
,
171
173
.