Mutation induced by DNA damage: a many protein affair

Mutation Research, 236 (1990) 301-311 DNA Repair Elsevier

MUTDNA 06013

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Mutation induced by DNA damage: a many protein affair

Harrison Echols a and Myron F. Goodman b Department of Molecular and Cell Biology, University of California, Berkeley, CA (U. S.A.) and b Department of Biological Science,

University of Southern California, Los Angeles, CA (U.S.A.)

(Accepted 12 March 1990)

Keywords: DNA damage, mutation induced by; DNA polymerase

The survival of an organism depends on the exceptionally precise duplication of its genome. To achieve this precision, the DNA polymerase itself must have extraordinary accuracy. In addition, a variety of repair mechanisms are needed to remove DNA lesions ahead of the replication fork and to correct mistakes left behind by the replication machinery. Mechanisms must also exist to deal with the potentially catastrophic encounter of a DNA polymerase with a replication- blocking lesion. In this brief review, we will consider the mechanisms responsible for the replication skills of the polymerase and the sources of occasional failure, with an emphasis on the SOS response induced by a replication-blocking lesion.

Under normal growth conditions, error frequencies in the duplication of the Escherichia coli genome are 10 - 9 to 10 -1° per base replicated. This high fidelity is achieved by a 3-step process: (1) the correct selection of the complementary deoxynucleoside triphosphate substrate during 5' --, 3' incorporation (base selection); (2) exo- nucleolytic 3' ~ 5' editing of a noncomplementary deoxynucleoside monophosphate misinserted at the end of the growing chain; (3) post-replicative mismatch repair. DNA polymerase III holoenzyme (pol III) is responsible for the majority of

Correspondence: Dr. Harrison Echols, Department of Molecu- lar and Cell Biology, University of California, Berkeley, CA (U.S.A.).

chromosomal replication in E. coli and is therefore probably the major determinant of the fidelity of genome duplication. Through base selection and editing, the polymerase itself provides about 106-107 to the overall fidelity. This accuracy is an astounding biochemical achievement because the correct (Watson-Crick) base pairs do not exhibit large structural or energetic differences from some incorrect base pairs ( e . g . G . T vs. A -T) . Even some of these 'spontaneous' mutations are likely to arise from DNA lesions (e.g. deamination of cytosine or depurination).

Mutagenic lesions in DNA fall into two general (but overlapping) classes: those that inhibit DNA replication (replication-blocking); and those that allow replication to proceed with diminished fidelity (error-promoting). The introduction of replication-blocking lesions into DNA induces the SOS response; as a consequence DNA replication is restored, and the lesions are converted functionally into error-promoting sites. Error-promoting lesions typically increase mispairing of bases during DNA replication (e.g. O6-methylguanine and 2-aminopurine). Replication-blocking lesions typically introduce substantial distortion into the DNA (e.g. pyrimidine dimer).

DNA polymerase III (and other polymerases) are finely tuned machines for precise and rapid replication of DNA. We believe that the extraordinary accuracy of pol III depends on an exquisite demand for the equivalent geometry of the Wat- son-Crick base pair. As a consequence, a substan-

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tial distortion in the pairing capacity of a base will block replication. Base insertion fidelity is designed to refuse such pairings, and the editing exonuclease will preferentially remove even a 'correctly' inserted base (e.g. A opposite template T in a cyclobutane dimer) because the distorted pair looks like a mismatch. The rescue of such a stalled polymerase is a major cellular operation. In the following sections, we will consider the response of D N A polymerases to mutagenic lesions and the role of the additional proteins - - RecA, UmuC and UmuD - - that are known to function in the mutagenic SOS response.

DNA polymerases

Fidelity mechanisms in replication. A molecular understanding of the source of replication errors requires a knowledge of the detailed mechanism by which D N A polymerases select the correct complementary base from the pool of possible mispairs. A combination of kinetic and structural work now in progress should ultimately yield this insight (Boosalis et al., 1987; Freemont et al., 1988; Kuchta et al., 1988; Sloane et al., 1988). Based on kinetic work with pol III, we have sug- gested a 'geometric model ' of base selection; the active site of the polymerase is designed to accept the geometrically identical Watson-Cr ick base pairs, A - T and G . C, and to reject those base pairs differing even slightly from this geometry (even the highly similar 'wobble ' G . T pair) (Sloane et al., 1988). This proposal is based on the observation that the most frequently misinserted bases are those that are likely to deviate least from Watson-Crick geometry, as judged by X-ray crystallography of mismatched oligonucleotides (Brown et al., 1986). A demand for Watson-Cr ick geometry at the transition state might be mani- fested either in more rapid dissociation of incorrect dNTP or slower phosphodiester bond formation for misoriented bases or more likely both (Goodman, 1988).

One common misinsertion error for many D N A polymerases in vitro (though not pol III) is A • A, which is not consistent with the geometric model (Boosalis et al., 1987). Opposite an apurinic site, the insertion of A is frequent in vivo and in vitro (Loeb and Preston, 1986). Possibly insertion of A

is a normally regulated 'default mechanism' by which a polymerase can traverse a template region lacking a capacity for geometric recognition. In addition to misinsertion errors, D N A polymerases make errors in template alignment, leading to frameshifts and 'dislocation' base substitution mutations (Kunkel and Alexander, 1986; Kunkel and Soni, 1988; Boosalis et al., 1989).

The second line of defense for replication errors is the editing function. At present, the most likely general discrimination mechanism for the editing exonuclease is the melting capacity of the mispaired D N A (Brutlag and Kornberg, 1972: Muzyczka et al., 1972: Petruska and Goodman, 1985). Structural studies seem to require the melting model for D N A polymerase I of E. coli because the exonuclease site will only accept single- stranded D N A (Freemont et al., 1988). The editing capacity of D N A polymerases is undoubtedly augmented by delayed chain elongation from a misinserted nucleotide (Petruska et al., 1988; Kuchta et al., 1988; Perrino and Loeb, 1989; Mendelman et al., 1990). Mismatch repair is a post-replicative error-correction mechanism that will not be considered here.

Error-promoting mutagenic events. Error-promoting mutagenic treatments are generally those that alter the precision of base-pairing recognition during replication. A common class of mutagens consists of analogues of normal nucleotides which exhibit ambiguous base pairing properties. For example, thymine analogues such as 5-bromouracil and 5-fluorouracil form normal base pairs with A, but also mispair with G more often than the normal T (Lasken and Goodman, 1984, 1985; Sowers et al., 1988, 1989). An adenine analogue 2-aminopurine pairs with T but can mispair with C (Watanabe and Goodman, 1981; Mhaskar and Goodman, 1984). These mispairs occur with the analogue present either as triphosphate substrates for insertion by polymerase or as template bases, stimulating both A • T --* G - C and G - C --* A . T transitions (Freese, 1959).

A variety of chemicals modify normal DNA bases and alter base-pairing specificities. N- Methyl -N ' -n i t ro-N-ni t rosoguanidine ( M N N G ) and N-methyl-N-ni t rosourea alkylate normal D N A bases and are toxic and mutagenic to cells.

When G is alkylated at the 6 position on the purine ring, the G . T mispair is stimulated strongly (Singer et al., 1989). In a similar manner, O4-MeT exhibits an increased probability to form mispairs with G (Dosanjh et al., 1990). There exist well characterized constitutive and inducible enzyme systems to rid the cells of alkylation damage (Lin- dahl et al., 1988).

Replication-blocking mutagenic events. As the term implies, certain DNA lesions effectively inhibit DNA replication. The damage site is presumably sufficiently distorted that the geometric recognition mechanism does not allow effective base insertion. In the extreme, the template base may be completely absent as in depurination events. In addition, at a highly distorted site even a correctly inserted base will look like a mismatch to the editing exonuclease. For certain cases, such as cyclobutane pyrimidine dimers, replication probably does not stop because the damaged base is incapable of base-pairing, but because the distorted base pair is unacceptable to the enzyme (Banerjee et al., 1988). The capacity of pyrimidine dimers and abasic sites to block DNA replication in vitro has been firmly established (Moore et al., 1981; Sagher and Strauss, 1983). More recent experiments have established the same point in vivo, most dearly by work with single-strand M13 viral DNA containing single cis-syn-cyclobutane dimers or abasic sites (Banerjee et aI., 1988; Lawrence et al., 1990). Some in vitro experiments with random pyrimidine dimers appear to indicate more frequent bypass than that noted in this in vivo work (Livneh, 1986); however, all experiments in vivo and in vitro concur that pyrimidine dimers are effective in blocking DNA replication in the absence of intervention by the SOS response.

The cellular response to a replication-blocking lesion is the SOS response (Radman, 1975; Witkin, 1976; Walker, 1984). As considered below, the SOS response depends on the activation of RecA for regulatory and probably direct biochemical functions, including the cleavage of the LexA repressor of SOS-induced genes. Replication re- sumes and mutations are produced. Most mutations appear to be targeted to the sites of DNA damage (Miller, 1982, 1985; Wood and Hutchin-

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son, 1984); in addition, there are untargeted mutagenic events in undamaged DNA (Caillet-Fauquet et al., 1984). A high mutation rate is evident when the SOS response is activated by a mutation in recA rather than by an external replication-blocking lesion (Witkin and Wermundsen, 1978). These mutations might represent untargeted events, but probably derive mainly from SOS processing of 'cryptic' lesions (such as apurinic sites) generated in DNA without external mutagenic treatment (Witkin, 1976; Miller and Low, 1984; Sweasy et al., 1990). Although the details are controversial, the most direct interpretation of the available data would suggest that SOS mutagenesis involves an induced alteration of DNA replication to a re- duced fidelity mode, allowing translesion DNA replication and targeted mutagenesis, as well as a lower level of untargeted events (Villani et al., 1978; Echols, 1982).

DNA polymerases in SOS. Which of the three E. coli DNA polymerases is involved in SOS mutagenesis? Certainly pol III is required. The involvement of pol III was originally deduced from indirect experiments (Bridges et al., 1976). More recently, a specially constructed tempera- ture-sensitive strain of pol III was used to show that pol III must be active for SOS mutagenesis (Hagensee et al., 1987).

Other recent experiments have indicated that pol II also might have a role in SOS events. Pol II activity is induced 7-fold by SOS induction, and this induction depends on inactivation of the LexA repressor (Bonner et al., 1988). The role of pol II is difficult to assess at present because the only known mutation was identified by an assay for the absence of pol II activity in vitro and lacks a phenotype in vivo (Hirota et al., 1972; Smith et al., 1976). However, the SOS inducibility of this enzyme makes it likely that there is a role for pol II in the SOS response. Although perhaps not active in translesion replication (based on the defined role for pol III), pol II might have an important function in DNA-repair events. Pol I has also been implicated in the SOS response by the observation of an altered form of the enzyme termed pol I* (Lackey et al., 1982). Pol I* has lowered fidelity, as judged by in vitro assays (Lackey et al., 1982). However, UV-induced muta-

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genesis appears to be normal in polA mutants (Witkin, 1970), including a recent deletion study (Bates et al., 1989). Because the pathways of replicative repair are multiple and complex, both pol I * and pol II may have special functions.

In summary, the SOS rescue of a stalled polymerase probably involves the interaction of SOS proteins with pol III. The biochemistry of translesion replication depends on understanding these interactions.

RecA protein

Multiple roles of RecA in the SOS response. The RecA protein of E. co# has been extensively characterized in its role in homologous genetic recombination. RecA forms a nucleoprotein filament on single-strand (SS) DNA in the presence of a mononucleotide co-factor (ATP, dATP or ATP-'t-S). In an ATP-consuming reaction, RecA then searches out homology in duplex (DS) DNA and catalyzes the branch migration reaction that generates heteroduplex DNA (Cox and Lehman, 1987). RecA also has several specialized roles designed to save the cellular soul in the SOS response. The multiple functions of RecA protein are summarized in the Table and described below.

RecA Activities H o m o l o g o u s R e c o m b i n a t i o n - - SS transfer T u r n o n S O S G e n e s - - L e x A cleavage 7 P o i n t M u t a t i o n s - - UmuD cleavage/Repl bypass I9/

• "! SOS R e p l i c a t i o n R e s t a r t - - Repl. bypass II? | D u p l i c a t i o n M u t a g e n e s i s - - DS transfer? J

RecA has two regulatory roles in the SOS response and probably three functionally distinct 'direct' biochemical functions. The regulatory functions are clearly defined biochemically: cleavage of the LexA repressor for SOS genes to turn on the induced response; and cleavage of the UmuD protein to activate the mutagenic pathway. The direct activities are much more speculative. RecA is likely to facilitate translesion mutagenic replication (replicative bypass I) and another replicative rescue operation that we have termed replication restart (replicative bypass II). In addition, RecA is required for another SOS-induced mutagenic pathway responsible for large duplications.

The first SOS function of RecA to be identified is proteolytic cleavage of k cI protein, allowing phage k to escape a potentially doomed cell (Craig and Roberts, 1980; Roberts and Devoret, 1983). The critical role for regulating induction of bacterial proteins is cleavage of the LexA repressor of SOS genes (Little and Mount, 1982). The cleavage reaction as initially defined requires SS DNA (polynucleotide cofactor) and a mononucleotide cofactor (Craig and Roberts, 1980; Lit- tle and Mount, 1982).

More recent experiments have indicated that association of RecA with damaged duplex DNA might constitute a second pathway to SOS induction. Purified RecA associates much more efficiently with UV-irradiated DS DNA than with nonirradiated DS DNA (Lu et al., 1986). The product of this reaction is a nucleoprotein filament covering a long stretch of DNA (Rosenberg and Echols, unpublished work). Presumably the DNA lesion serves as a 'micro single-stranded' region that provides a nucleation site for RecA to initiate cooperative binding on DS DNA (Echols, 1982; Lu et al., 1986). RecA also mediates cleavage of LexA after association with DS DNA (Lu et al., 1986; Lu and Echols, 1987). Thus, binding to the lesion in DS DNA is likely to serve as an inducing signal (presumably in addition to the SS DNA activation, although the in vivo route remains to be established). A number of older in vivo experiments indicated two pathways to an 'inducing signal' (summarized in McPartland et al., 1980). Additional work has supported the role of RecA and DS DNA. Certain recA gene mutations lead to 'constitutive' activation of the SOS response (no lesion needed) and constitutive expression of the UmuCD pathway for point mutations (Witkin, 1976; Waller, 1984). One of these mutant RecA proteins (RecA441) binds efficiently to DS DNA with no lesions and becomes active for cleavage of LexA; this lesion-independent DS binding can account for constitutive expression of SOS genes (Lu and Echols, 1987).

To summarize the induction pathway, the 'inducing signal' in vivo for regulated cleavage of LexA is probably the association of RecA with a polynucleotide cofactor (SS DNA or DS DNA). The cleavage reaction also requires a mononucleotide cofactor; this molecule might be ATP,

dATP, or a tight-binding cofactor analogous to ATP-v-S, which works best in vitro. Full SOS induction probably requires blocked replication as well as damaged DNA (Ennis et al., 1985). The replication block might serve to generate an effi- cient cofactor activity as a consequence of an 'idling' reaction by the polymerase (e.g. higher concentration of dATP or a tight-binding cofactor).

In addition to RecA, SOS mutagenesis requires the UmuC and UmuD proteins, synthesis of which is induced by cleavage of LexA (Walker, 1984). Recent work has revealed another regulatory role of RecA in the SOS response - - cleavage of UmuD to a processed product, UmuD' , which is probably the active form for mutagenesis. Se- quence homology between LexA and UmuD near the LexA cleavage site had been noted (Perry et al., 1985). The purification of UmuD from an engineered overproduction system allowed a direct test; RecA carries out cleavage of UmuD (Burck- hardt et al., 1988). A mutant RecA protein defective in mutagenesis, RecA430, fails to cleave UmuD. UmuD is also cleaved in vivo (Shinagawa et al., 1988); cleavage occurs at the site expected from homology with LexA (Woodgate et al., 1989). The biological importance of this cleavage reaction has been demonstrated by the engineered synthesis of UmuD' in vivo. Production of UmuD' in vivo is sufficient for SOS mutagenesis and bypasses the defect of recA430 bacteria for mutagenesis (Nohmi et al., 1988). Thus, RecA-mediated cleavage of UmuD to UmuD' is required for mutation by the UmuCD pathway.

An interesting aspect of the protein cleavage reactions mediated by RecA is the capacity of those proteins to self-cleave at the same site at alkaline pH (Little, 1984; Slilaty and Little, 1987; Burckhardt et al., 1988). The single scissile peptide bond in each protein probably undergoes hydroly- sis by a mechanism similar to that of serine pro- teases (Slilaty and Little, 1987). Thus all three proteins carry a homologous 'self-destruct' do- main, for which the reactivity is greatly accel- erated by RecA. The UmuD reaction contrasts with LexA in that cleavage by both pathways is slower for UmuD; this differential cleavability presumably allows SOS induction to occur at low concentrations of RecA, but defers the mutagenic

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pathway for a 'last resort' function (Burckhardt et al., 1988).

A 'direct' biochemical role for RecA in mutagenesis had been inferred from the observation that mutational inactivation of LexA did not allow SOS mutagenesis (Blanco et al., 1982; Witkin and Kogoma, 1984; Ennis et al., 1985). The dis- covery of the UmuD cleavage reaction initially left open the possibility that the inferred 'direct' role of RecA in translesion DNA replication was production of UmuD' . However, recent genetic ex- perirnents have indicated strongly that RecA does have an additional role in SOS mutagenesis beside cleavage of LexA and UmuD, presumably an activity at the replication-blocking lesion (Nohmi et al., 1988; Dutriex et al., 1989; Sweasy et al., 1990). In the Table, we have called this activity 'replicative bypass I'.

What is the direct role of RecA in mutagenesis? At this point, we can make reasonable guesses, but the answer awaits a biochemical reconstitution of translesion replication. Several years ago, we speculated that RecA might facilitate translesion DNA replication (and thereby mutagenesis) by binding to the lesion site in DS DNA; RecA would then be 'prepositioned' to interact with the incoming polymerase, relaxing its specificity and providing for replication past the lesion with re- duced fidelity (Echols, 1982). The ability of RecA to associate preferentially with lesion-containing DNA, as noted above, is consistent with this idea. One possible function of RecA that might help pol III at the replication-blocking lesion is inhibition of the exonuclease activity of the polymerase. Even if the polymerase manages to insert a correct base opposite a lesion, the inserted base will probably be recognized as a mispair by the editing function because of the distorted configuration. In support of this idea, RecA inhibits the exonuclease of pol III on a mispaired substrate and also the exonuclease of the isolated c subunit (Fersht and Knill-Jones, 1983; Lu et al., 1986). RecA will also stretch the single-stranded template DNA strand (Cox and Lehman, 1987); this process might facilitate base insertion at the distorted lesion site (Lu and Echols, 1987).

Recent experiments in vivo have been interpre- ted in terms of a role for inhibition of editing in SOS mutagenesis. Overproduction of c from a

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multi-copy plasmid serves to inhibit SOS mutagenesis (Jonczyk et al., 1988; Foster et al., 1989). However, the interpretation of these experiments is complicated because the overproduced ~ might not be functioning as part of the polymerase. Nevertheless, even if free ~ can interfere with translesion replication, these experiments are consistent with a need for an inhibition of E activity in the normal SOS process.

In addition to the UmuCD pathway for point mutations, RecA participates in an additional SOS rescue operation. After UV-irradiation, DNA synthesis is severely inhibited, but recovers after a few minutes in an SOS-induced response (Khidhir et al., 1985; Witkin et al., 1987). This repfication restart does not occur in cells carrying completely defective mutations in the recA gene, but occurs normally in recA + umuC bacteria. More inter- estingly, replication-restart fails to occur in certain recombination-proficient mutants in the recA gene, for example the recA430 described above (Witkin et al., 1987; Dimpfl and Echols, unpublished work). The replication-restart function of RecA is probably much more important to cellular survival than the UmuCD pathway; mutants blocked in replication-restart are extremely UV-sensitive, much more so than umuC- or umuD- mutants (Witkin et al., 1987). Because of its independence of U m u C / D , in the Table we have assigned replication-restart to a 'replicative bypass II' pathway.

Recent efforts to explore the scope of the SOS response have revealed yet another SOS function of RecA - - induced formation of large duplications. Tandem gene duplications are an important type of mutation in E. coli, occurring at high frequency (10 -5 to 10 -3 per cell) and often including large segments of the chromosome (Anderson and Roth, 1977; Petes and Hill, 1988). These frequent large duplications are primarily RecA-dependent events, with endpoints corre- sponding to regions of homology. The frequency of duplications rises about 10-fold when a wild- type recA gene is replaced with the recA 730 allele that confers a constitutive SOS response (Dimpfl and Echols, 1989). Enhanced duplication formation does not depend on an active umuC gene; however, an active recF gene is required, indicat- ing that duplication formation involves the RecF pathway of recombination, rather than the RecBC

pathway. Thus, the induced pathway for duplication mutations appears to be distinct from the UmuCD pathway for point mutations and also differs from the major normal recombination pathway, RecBC. Possibly the duplication pathway (normally rare) becomes elevated when RecA efficiently forms nucleoprotein filaments on DS DNA (Dimpfl and Echols, 1989).

Replicative bypass of DNA lesions

The UmuC and UmuD mutagenesis proteins. The UmuC and UmuD proteins are required specifically for SOS mutagenesis (Kato and Shinoura, 1977; Steinborn, 1978; Bagg et al., 1981). Therefore an understanding of the mutagenic pathway clearly requires a knowledge of the biochemical activities of those proteins. As noted above, the functionally active form of UmuD is the cleaved form, UmuD' . Recently UmuD' and UmuC have been purified (Woodgate et al., 1989). As judged by immunoprecipitation experiments in crude extracts, UmuC associates with UmuD' in vivo. UmuC and U m u D ' also associate tightly in vitro, based on cosedimentation experiments. Thus the likely mutagenic component is the U m u C - U m u D ' complex (Woodgate et al., 1989). How- ever, the biochemical function of U m u C - U m u D ' remains to be established.

Possible mechanisms for SOS-mediated rescue of replication. One possible pathway for translesion replication and replication-restart is shown in Fig. 1 (Woodgate et al., 1989). This model incorporates current knowledge in a general way, leaving open the unknown biochemical mechanisms. We pre- sume that translesion replication (replicative bypass I) is facilitated by formation of a lesion- localized nucleoprotein structure ( 'mutasome') involving RecA, U m u C - U m u D ' and pol III. Simi- lar site-specific multiprotein complexes localize and control the reactivity of many DNA transactions (Echols, 1986). As noted above, the presence of RecA might help in two ways: alter the distorted template conformation to one more palat- able for pol III; and inhibit the editing exonuclease. U m u C - U m u D ' might facilitate DNA-chain elongation by keeping the polymerase engaged in bypass attempts (either preventing dis-

sociation or favoring reassociation) (Bridges and Woodgate, 1985; Lu and Echols, 1987; Hevroni and Livneh, 1988; Battista et al., 1990). The assembly of the mutasome might begin with a RecA nucleoprotein filament at the site of the lesion (Fig. 1). UmuD will be cleaved to UmuD' either at this site or another lesion site, and the UmuC- UmuD' complex will be available to facilitate a productive encounter of pol III with the site of DNA damage. UmuC-UmuD' might be brought to the mutasome by association with RecA (Frei- tag and McEntee, 1989; Sweasy et al., 1990), or by interaction with pol III (or both). UmuC is a highly basic protein, a property likely to facilitate association of the UmuC-UmuD' complex with

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DNA. An alternative pathway for assembly of the same nucleoprotein structure would be created by RecA binding to SS D N A generated downstream from the lesion after the polymerase stalls (Ro- berts and Devoret, 1983) (bottom of Fig. 1).

The nucleoprotein assembly at the bottom of Fig. 1 might also mediate the replication-restart reaction (replicative bypass II). The RecA-coated SS DNA downstream from the blocked polymerase will presumably associate with the replicated duplex from the complementary strand because this interaction is equivalent to the initial step in formation of heteroduplex DNA in general recombination. In the resultant 3-stranded structure, pol III might bypass the lesion by copying

f ~C,

I I I I I I t l l l l l

UmuC/D

© RecA

f

Cleave UmuD ~ Cleave LexA (mutagenesis) (induce SOS)

UmuC/D' Pol I11

I I I l l l l l l

Translesion replication

Replication restart (strand switch?)

Fig. 1. Possible mechanism of SOS-induced mutagenesis. A multiprotein complex is assembled at the DNA lesion, including UmuC, UmuD', RecA and DNA polymerase III holoenzyme. This nucleoprotein aggregate provides for replication across the lesion with the introduction of mutations. A possible alternative pathway for error-free replicative bypass is DNA synthesis with the daughter strand

as template in a three-stranded complex generated by RecA.

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the undamaged daughter strand instead of the blocked parental strand, switching back after cleating the lesion (Echols, 1982; Woodgate et al., 1989). UmuC-UmuD' would not normally be required for this reaction. However, there is one mutant RecA for which replication restart depends on UmuC-UmuD' (Witkin et al., 1987). This observation indicates that translesion replication and replication-restart are related pathways. The proposed replication-restart pathway would augment and might sometimes initiate the previously defined recombinational repair pathway (Rupp et al., 1970).

To summarize, the RecA-facilitated pathways of translesion replication and replication restart clearly involve fascinating alterations in the biochemistry of DNA replication, mediated by RecA and UmuC-UmuD' (and perhaps other proteins). However, the biochemical mechanisms remain to be established.

Some biological comments

The enhanced frequency of point mutations associated with SOS induction can be considered from two (not necessarily exclusive) points of view. The UmuCD mutagenic pathway is an important component of an induced cellular survival mechanism, and mutations are a 'side-effect'; or, the UmuCD pathway exists as a population survival mechanism that enhances genetic variation, in- creasing the chances for a fortunate variant adapted to survive the environmental stress (Rad- man, 1980; Echols, 1981; Echols, 1982). The UmuCD pathway contributes essentially all of the mutations and also contributes to survival, but to a small extent compared to error-free mechanisms. The genetic variation hypothesis suggests that SOS mutagenesis might be expected to include other types of mutations, such as duplications and re- arrangements, which might have profound pheno- typic effects: The recent finding of an SOS inducible pathway for large tandem duplications dem- onstrates that SOS produces a broad mutagenic response. In this sense, SOS mutagenesis might be considered an example of 'inducible evolution' (considered in more detail in Echols, 1981, 1982).

In other recent experiments of notable evolu- tionary impact, clear evidence has been presented

for mechanisms that accelerate the appearance of adaptively favorable mutations under conditions of extreme starvation (Cairns et al., 1988; Hall, 1988). This work has led to the fascinating sugges- tion that the favorable mutations are somehow directed to the appropriate gene. However, the fact that SOS mutagenesis is a broad spectrum inducible response involving different types of mutations and pathways leaves open the possibility that a similar response might be operative in these systems, followed by selection of the fortunate variant. Certain classes of mutations might increase in response to specific stress signals, rather than to a needy locus.

In conclusion, new ideas and new data have given us a view of mutation as a biochemically complex and highly regulated event. Although there are vast gaps in our knowledge of biochemical mechanism and biological impact, we can foresee a period of rapid progress in both realms.

Acknowledgments

We thank Malini Rajagopalan, Marcie Rosen- berg and Evelyn Witkin for comments on the manuscript and Richard Eisner for editorial help. Work from the authors' laboratory has been supported by grants CA41655 and GM21422.

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Mutation induced by DNA damage: a many protein affair