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32 Sandler, S.J. et al. (1996) Differential suppression of 39 Capaldo, F.N. et al. (1971) Analysis of the growth of 45 Kim, S. et al. (1996) ␶ protects ␤ in the leading-strand priA2::kan phenotypes in Escherichia coli K-12 by recombination-deficient strains of Escherichia coli K-12. complex at the replication fork. J. Biol. mutations in priA, lexA, and dnaC. Genetics 143, 5–13 J. Bacteriol. 118, 242–249 Chem. 271, 4315–4318 33 Zavitz, K.H. and Marians, K.J. (1992) ATPase-deficient 40 Learn, B.A. et al. (1997) Cryptic single-stranded-DNA 46 Yuzhakov, A. et al. (1996) assembly reveals mutants of the Escherichia coli DNA replication protein binding activities of the phage lambda P and the basis for asymmetric function in leading and PriA are capable of catalysing the assembly of active Escherichia coli DnaC replication initiation lagging strand replication. Cell 86, 877–886 primosomes. J. Biol. Chem. 267, 6933–6940 proteins facilitate the transfer of E. coli DnaB 47 Xu, L. and Marians, K.J. Purification and characterization 34 McGlynn, P. et al. (1997) The DNA replication protein onto DNA. Proc. Natl. Acad. Sci. U.S.A. 94, of DnaC810: a primosomal protein capable of PriA and the recombination protein RecG bind D-loops. 1154–1159 bypassing PriA function. J. Biol. Chem. (in press) J. Mol. Biol. 270, 212–221 41 Fuller, R.S. and Kornberg, A. (1983) Purified dnaA 48 Sandler, S.J. et al. (1999) dnaC mutations suppress 35 Nurse, P. et al. (1999) Two modes of PriA binding to protein in initiation of replication at the Escherichia coli defects in DNA replication- and recombination-associated DNA. J. Biol. Chem. 274, 25026–25032 chromosomal . Proc. Natl. Acad. functions in priB and priC double mutants in 36 Liu, J. et al. (1999) Replication fork assembly at Sci. U.S.A. 80, 5817–5821 Escherichia coli K-12. Mol. Microbiol. 34, 91–101 recombination intermediates is required for bacterial 42 Marszalek, J. and Kaguni, J.M. (1994) DnaA protein 49 Seigneur, M. et al. (1998) RuvAB acts at arrested growth. Proc. Natl. Acad. Sci. U. S. A. 96, directs the binding of DnaB protein in initiation of DNA replication forks. Cell 95, 419–430 3552–3555 replication in Escherichia coli. J. Biol. Chem. 269, 50 Scott, J.F. et al. (1977) A mechanism of duplex DNA 37 Jones, J.M. and Nakai, H. (1997) The ␾X174-type 4883–4890 replication revealed by enzymatic studies of phage promotes replisome assembly at the site 43 Hiasa, H. and Marians, K.J. (1994) couples ␾X174: catalytic strand separation in advance of of recombination in bacteriophage Mu transposition. leading- and lagging-strand DNA synthesis from oriC. replication. Proc. Natl. Acad. Sci. U. S. A. 74, 193–197 EMBO J. 16, 6886–6895 J. Biol. Chem. 269, 6058–6063 51 Sandler, S.J. and Marians, K.J. (2000) Role of PriA in 38 Courcelle, J. et al. (1999) Recovery of DNA replication 44 Kim, S. et al. (1996) Coupling of a replicative replication fork reactivation in Escherichia coli. in UV-irradiated Escherichia coli requires both excision polymerase and helicase: a ␶-DnaB interaction J. Bacteriol. 182, 9–13 repair and RecF protein function. J. Bacteriol. 181, mediates rapid replication fork movement. Cell 84, 52 Haber, J.E. (1999) DNA recombination: the replication 916–922 643–650 connection. Trends Biochem. Sci. 24, 271–275

umuD, must be present to observe mutagenesis above spontaneous back- ground levels1 (the term ‘Umu’ refers to Coping with replication UV mutagenesis). UmuC and UmuDЈ, the shortened form of UmuD required for ‘train wrecks’ in Escherichia SOS mutagenesis1, produce a tightly 2 Ј bound complex , UmuD 2C, that has re- cently been shown to contain an intrin- coli using Pol V, Pol II and sic, low-fidelity DNA polymerase activity, E. coli Pol V (Ref. 3–5). Pol V (or UmuDЈ C) catalyses template-directed RecA proteins 2 nucleotide incorporation in the absence of any other and, when act- ing in conjunction with RecA protein, is Myron F. Goodman responsible for SOS-induced mutagen- esis as a result of error-prone repair. DNA replication machineries tend to stall when confronted with damaged DNA This review focuses on recent bio- template sites, causing the biochemical equivalent of a major ‘train wreck’. chemical and genetic discoveries that A newly discovered bacterial DNA polymerase, Escherichia coli Pol V, acting in have shed light on how two SOS-induced conjunction with the RecA protein, can exchange places with the stalled replica- polymerases, the newly discovered tive Pol III core and catalyse ‘error-prone’ translesion synthesis. In contrast to Pol V and the enigmatic Pol II, can copy Pol V-catalysed ‘brute-force, sloppier copying’, another SOS-induced DNA polym- damaged DNA templates: either by Pol V- erase, Pol II, plays a pivotal role in an ‘error-free’, replication-restart DNA repair catalysed copying of a lesion (trans- pathway and probably involves RecA-mediated homologous recombination. lesion synthesis) or by Pol II-catalysed reinitiation of replication6 downstream from the lesion, a process called in- REPLICATIVE DNA POLYMERASES ‘ex- (abasic site) can cause problems akin to duced replisome reactivation7,8 or repli- pect’ to encounter few, if any, serious a major ‘train wreck’, by derailing of the cation restart9. An important distinction impediments when duplicating DNA at replication complex. Leading- and lagging- between the two pathways is that Pol II- rates approaching 1000 nt per sec in strand replication complexes are likely to catalysed replication restart is error- prokaryotic cells. Although polymerase uncouple when blocked at a template free, whereas Pol V translesion synthe- pause sites might arise from secondary damage site, causing dissociation of a sis is error-prone (i.e. mutagenic). Both structures or from ongoing RNA transcrip- polymerase and its subunits. pathways require the action of RecA tion, these cause no serious delay in How do cells manage to survive in the protein, albeit in two different roles. replication fork movement. However, this presence of heavily damaged DNA? An is not so for DNA damage. The presence acronym, ‘SOS error-prone repair’, has Genetic and biochemical basis of of a UV-induced pyrimidine photodimer been coined to describe a variety of bio- SOS-induced mutagenesis in E. coli or the loss of just a single template base chemical reactions aimed at alleviating UV radiation is injurious to cells, result- the ravages of DNA damage, causing the ing primarily in the appearance of cyclo- induction of at least 30 different genes in butane dimers and 6-4 pyrimidine– M.F. Goodman is in the Dept of Biological 1 Sciences and Chemistry, University of Escherichia coli . Many of these genes are pyrimidone photoproducts at sites of Southern California, University Park, required for excision and recombination- adjacent pyrimidine bases in DNA Los Angeles, CA 90089-1340, USA. dependent repair of DNA damage, whereas (Fig. 1). When such lesions are encoun- Email: [email protected] two key SOS-induced genes, umuC and tered by DNA polymerases, replication 0968 – 0004/00/$ – See front matter © 2000, Elsevier Science Ltd. All rights reserved. PII: S0968-0004(00)01564-4 189 REVIEWS TIBS 25 – APRIL 2000

exogenous Pol III core3. We suggested, (a) (b) (c) Ј therefore, that UmuD 2C might harbor O O an intrinsic low-fidelity DNA polymerase CH3 H 4 O activity that could be used to copy past O CH N 3 5 OH H N CH3 2 4 damaged DNA template sites at which O H N 3 5 CH3 Pol III replication complexes become 2 6 3 1 N H stalled . Additional data supporting this O H 4 3 O O N 2 N 6 interpretation demonstrated that high 5 H 2 O 1 nucleotide misincorporation levels also O H3C 1 N 6 N occurred opposite undamaged DNA tem- H plate sites3. Indeed, earlier genetic data Ti BS suggested a role for the Umu proteins in causing both targeted and ‘untargeted’ Figure 1 point mutations15. However, in contrast Structures depicting DNA template damage sites used to measure DNA polymerase V to our data, Livneh and colleagues re- mutasome (Pol V Mut)-catalysed translesion synthesis in vitro. (a) Synthetic abasic lesion ported no detectable abasic lesion by- (tetrahydrofuran moiety). (b) 6-4 T–T pyrimidine–pyrimidone photoproduct. (c) cis-syn T–T cyclobutane dimer. pass, unless Pol III HE was present along with the Umu proteins in their assay20. There was an important caveat con- is slowed down, although not entirely Although highly purified RecA and cerning Umu-catalysed DNA synthesis Ј blocked. Mutations are targeted directly Pol III HE were readily available, the in- because of the possibility that UmuD 2C opposite the lesion sites10,11, demon- herent insolubility of UmuC (Ref. 16) might be contaminated with small strating that translesion synthesis is an imposed a daunting obstacle in the suc- amounts of either Pol III core or Pol II error-prone process requiring the low- cessful reconstitution of SOS translesion (Ref. 3). Although the patterns of Ј fidelity Pol V. Targeted SOS-mutation synthesis. Harrison Echols and co- UmuD 2C-catalysed DNA synthesis on rates are increased by as much as sev- workers17 constructed the first success- damaged and undamaged DNA tem- eral 100-fold above spontaneous levels. ful lesion bypass assay by isolating in- plates were entirely unlike those ob- A key component required for SOS soluble UmuC from inclusion bodies, served for the three known E. coli DNA mutagenesis is RecA, a multifunctional solubilizing it in urea, purifying the de- polymerases, it was still possible that protein required for homologous recombi- natured protein, and then attempting to the properties of a contaminant polym- nation, induction of the SOS response renature UmuC by removing the de- erase might be grossly altered in the 1 Ј and SOS mutagenesis . RecA serves as a naturant. This process was successful presence of the UmuD 2C complex. In coprotease in the cleavage of the LexA insofar as an increase in translesion syn- fact, this was precisely the idea in the repressor protein to turn on the SOS re- thesis was observed when purified UmuC SOS mutagenesis model proposed by sponse. RecA plays a similar role in the was added to UmuDЈ, RecA and Pol III HE. Bryn Bridges and colleagues21. They cleavage of mutagenically inactive UmuD But the denaturation–renaturation pro- posited that unaided, Pol III HE is able to to UmuDЈ to turn on SOS mutagenesis. cess proved tenuous because the yields incorporate a nucleotide opposite the These reactions require the presence of of ‘active’ UmuC were both miniscule 3ЈT of a cis-syn T–T dimer, but that the activated RecA (RecA*), whereby RecA and variable18. Umu proteins, in the presence of RecA*, protein binds cooperatively to single- We used a different approach in purify- are then required to allow further exten- stranded DNA forming a RecA* filament ing the Umu proteins. Roger Woodgate sion to the 5ЈT site and beyond. Several in the presence of a mononucleotide co- designed a system in which UmuC and possibilities were proposed for this re- factor, ATP or dATP. However, genetic data UmuD were deleted from the E. coli action: for example, a direct effect of Umu have revealed that the RecA protein also chromosome and UmuC and UmuDЈ were on the efficiency of aberrant primer ex- plays a direct role in SOS mutagenesis, overexpressed from a high-copy-number tension, aided perhaps by a reduction in presumably through an interaction with plasmid19. We were then able to isolate 3Ј-exonuclease proofreading by the Pol III Ј 12–14 Ј UmuD 2C at a template lesion site . A substantial quantities of native UmuD 2C core in the presence of RecA protein. thorough review of the data and ideas in a tightly bound mutagenically active ؅ relating to SOS mutagenesis, but prior to complex that remained soluble in aque- UmuD 2C is a novel DNA polymerase, discovery of the roles played by Pol V ous solution throughout purification2. E. coli Pol V and Pol II, is present in Ref. 15. Z. Livneh’s group20, taking a different Several lines of evidence support our Ј approach, obtained UmuC as a maltose- original contention that UmuD 2C is a E. coli Pol V, with help from RecA, catalyses binding protein fusion (MBP–UmuC) to bona fide DNA polymerase, E. coli Pol V Ј mutagenic translesion synthesis which UmuD is added later. Both ap- (Ref. 4). Purification of UmuD 2C was An essential goal is to develop an proaches were successful, allowing re- carried out using an E. coli mutant con- in vitro biochemical assay using puri- constitution of an in vitro system in which taining a temperature-sensitive allele of fied proteins that mimics SOS muta- robust bypass of an abasic template le- DNA polymerase III [dnaE1026(Ts)] and ⌬ Ј genesis observed in vivo. Thanks to the sion occurred in the presence of UmuC, also lacking Pol II ( polB). The UmuD 2C thorough genetic analysis for SOS- UmuDЈ, RecA*, Pol III HE and E. coli single- complex by itself was found to catalyse induced mutagenesis, virtually all of the stranded-DNA-binding protein (SSB)3,20. nucleotide incorporation at undamaged important ‘players’ have been identi- However, our translesion synthesis DNA template sites. Even so, the Umu fied: the UmuDЈC proteins, RecA* and data revealed an unexpected finding, complex alone failed to catalyse incorpo- the replicative DNA polymerase III namely that a strong translesion synthe- ration opposite an abasic template lesion, holoenzyme (Pol III HE). sis signal occurred in the absence of the nor could it bypass the lesion. Translesion 190 TIBS 25 – APRIL 2000 REVIEWS synthesis required the simultaneous Ј ␤ presence of UmuD 2C, RecA, sliding Pol III HE ␥ γ complex clamp, clamp-loader complex and SSB β clamp (Ref. 4). The ␤,␥ complex comprises ′ 5′ subunits of the E. coli Pol III HE and are 3 X required to attain highly processive DNA Pol III core synthesis22. The five-subunit ␥ complex loads the ␤ circular clamp onto DNA, allowing it to tether the Pol III core to RecA* DNA during chain elongation. The ability to carry out translesion 3′ X 5′ synthesis reaction was essentially lost when any one of the above proteins was omitted from the reaction. Translesion ′ synthesis was also abolished when a Pol V (UmuD 2C) complex containing a mutant UmuC Ј (UmuD 2C104) was used, demonstrating Ј that UmuD 2C contains an intrinsic, error-prone DNA polymerase activity4. 3′ X 5′ However, because synthesis by Pol V ex- 5′ hibits low processivity, genetic data im- plying an absolute requirement for Pol V Mut Pol III in SOS mutagenesis23,24 are likely to reflect the need for a highly process- Pol V Mut ive replicative polymerase to complete chromosomal replication on undamaged 3′ X 5′ DNA, once lesion bypass has occurred. In A contrast, the initial nucleotide incorpo- ration and translesion synthesis events require the presence of RecA protein and the processivity factors, the ␤,␥ complex, of the holoenzyme interacting with Pol V instead of the Pol III core. 3′ X 5′ As Pol V must also interact with other A proteins to carry out translesion synthe- Pol III HE sis, we will use the term Pol V ‘muta- Ti BS some’ (Pol V Mut) to define this more extensive interacting system, comprising Figure 2 Pol V, RecA*, the ␤,␥ complex and SSB. Model describing DNA polymerase V mutasome (Pol V Mut)-catalysed error-prone trans- In analogy with the term ‘replisome’ to lesion synthesis. The initial lesion-bypass step involves dissociation of the replicative Pol III describe the Pol III HE system, the term core proximal to the DNA damage site (an abasic lesion X, shown on the leading strand). The presence of an abasic lesion effectively blocks further synthesis by the Pol III holo- ‘mutasome’ was coined by Hatch Echols enzyme (HE) (yellow), whereas Pol V Mut catalyses abasic translesion synthesis with at to describe the protein components re- least 100-fold higher efficiency, compared with the replicative Pol III4,25. Continued unwind- quired to copy DNA damage sites9. A ing of the DNA ahead of a stalled replication fork could enable formation of an activated Ј model depicting Pol V Mut-catalysed RecA nucleoprotein filament (RecA*; light blue). Binding of Pol V (UmuD 2C; dark blue and translesion synthesis based on current red) takes place at the 3Ј-OH vacated by Pol III core. It has been proposed that Pol V is tar- 49 in vitro data is shown in Fig. 2. geted to X by the presence of the RecA* filament end proximal to the lesion . Pol V-catal- The MBP–UmuC fusion protein has re- ysed translesion synthesis proceeds favoring the incorporation of A opposite X and requires the presence of RecA, ␤ sliding clamp, ␥ clamp-loading complex (␥ complex is required for load- cently been found to have an intrinsic ing the ␤ clamp on the DNA, but it is currently unknown if it remains bound to the DNA dur- polymerase activity5. In contrast to our ing translesion synthesis) and E. coli single-stranded-DNA-binding protein (SSB; not shown). data, Livneh and co-workers reported This combination of mutasomal proteins is denoted by Pol V Mut. Synthesis by Pol V has that the presence of the ␤,␥ complex was low processivity in the presence of RecA. Pol V dissociates following translesion synthesis not required for translesion synthesis. We and processive replication resumes using Pol III HE. have recently found that the ␤,␥ com- plex can be dispensed with when non- hydrolysable ATP␥S replaces ATP in the high to maintain an intact RecA filament. stimulated in the presence of RecA by a translesion synthesis reaction25, suggest- Thus, a putative role for RecA protein is remarkable 15 000-fold25. ing that RecA filament disassembly, oc- the activation of Pol V, while targeting it The polymerase activity of Pol V re- curring in the presence of ATP, but not to a DNA damage site, whereas the ␤,␥ sides in the UmuC protein4,5. However, ␥ Ј ATP S (Ref. 26), extinguishes Pol V ac- complex might help tether Pol V at the we favor designating UmuD 2C as Pol V tivity. There is one RecA protein per 3Ј-primer end during the RecA assembly– rather than UmuC for the following rea- 5–15 nt in our experiment, compared with disassembly process catalysed by ATP. sons. First, biochemical data suggest that Ј five RecA/nt in Livneh’s experiment; the The nucleotide incorporation activity of the native UmuD 2C forms a tight com- latter RecA level is probably sufficiently Pol V on undamaged DNA templates is plex following SOS induction enabling it 191 REVIEWS TIBS 25 – APRIL 2000

to be purified in a soluble form2. Native A over G, at both positions of a cis-syn that this error-prone polymerase is also UmuC by itself is insoluble in aqueous T–T photodimer by about 50-fold25. responsible for SOS-induced untargeted solution16. Second, the induced levels of In contrast with abasic and cis-syn mutations3,31. UmuD are roughly tenfold higher than T–T adducts, Pol V Mut has a different UmuC, rendering it unlikely that UmuC specificity of incorporation at the two A pivotal role for enigmatic E. coli is present by itself in vivo27. We further positions of a 6-4 T–T photoproduct. DNA polymerase II in SOS error-free note that the complex composed of the The mutasome favors incorporation of replication restart ␣, ⑀ and ␪ subunits is designated as the G over A opposite the 3ЈT by approxi- Although E. coli DNA polymerase II E. coli Pol III core despite the fact that mately 6:1, whereas it favors incorpo- was discovered in 1970 (Ref. 32), its the polymerase activity is confined to ration of A over G at the 5ЈT lesion site function in the cell has remained a mys- the ␣ subunit. by approximately 8:125. These data tery until recently6. A Pol II-null mutant agree with nucleotide incorporation (⌬polB) showed no obvious cellular A comparison of Pol V-catalysed frequencies deduced from in vivo mu- phenotype until coupled with chromo- nucleotide incorporation specificities with tational data10,11,29,30. In contrast, Pol III somal deletions of umuC and umuD mutational data HE favors incorporation of A at all of the (⌬umuCD). A combined Pol II UmuDC- A key test of relevance for the in vitro lesion sites described above25. A second null mutant (⌬umuCD ⌬polB) showed SOS model system is to demonstrate its important distinction between these en- decreased survival to irradiation with consistency with in vivo mutagenesis zymes is that Pol V Mut copies the three UV light, compared with either of the data. So far, three types of DNA damage lesions with considerably higher effi- single mutants, whereas survival of the sites have been copied using the Pol V ciency than Pol III HE (Ref. 25). The close ⌬polB single mutant was indistinguishable mutasome, an abasic moiety, a 6-4 T–T agreement between in vitro and in vivo from wild type6. The increased UV sensi- photoadduct and a cis-syn T–T photo- data, especially the generation of T→C tivity of the ⌬umuCD ⌬polB double mu- dimer (Fig. 1). Incorporation of A oppo- transition mutations at the 3ЈT of a 6–4 tant suggested that the Pol II and Umu site abasic lesions is favored in E. coli, a photoproduct, suggests that Pol V Mut, proteins might complement one another result commonly referred to as the ‘A and not Pol III HE modulated by Pol V in different UV repair pathways. rule’28. In agreement with the A rule, we Mut, is responsible for generating SOS- An in vivo assay using 3HdT incorpo- have found that Pol V Mut favors incor- induced lesion-targeted mutations. We ration into DNA to measure replication poration of A by about twofold over G, have recently found that Pol V Mut fidelity restart in UV-irradiated cells revealed with incorporation of pyrimidines oc- when copying undamaged template sites that DNA replication in wild-type cells curring much less frequently3. Pol V is typically in the order of 10Ϫ3–10Ϫ4 for was inhibited transiently, immediately Mut was also observed to incorporate almost all types of mispairs25, suggesting following exposure to UV light, but that 192 TIBS 25 – APRIL 2000 REVIEWS

DNA synthesis recovered rapidly within ~1–2 min7,8. In contrast, the resumption (a) γ complex of DNA synthesis was delayed by ~50 β clamp min when Pol II was absent6. A similar 3′ T T Pol III HE stalling result was also observed in a priA mu- disassembly tant background. The appearance of Ј Pol III UmuD also occurs at ~50 min following core SOS induction33, suggesting that the re- sumption of replication in the absence SOS induction of either Pol II or PriA proteins might be Ј attributable to Pol V Mut (UmuD 2C)- catalysed translesion synthesis. Support (b) 5′ for this idea was obtained using a 3′ D-loop Restart-primosome ⌬umuDC ⌬polB double mutant where replication restart was delayed for Pol II 100–110 min, at which time a ‘last resort’ T T recombination–repair pathway presum- ′ ably takes over6. The existence of two 3 distinct temporally-spaced pathways in replication restart, a umuDC-independent Pol III pathway, followed by one that is umuDC- Pol II dependent, has also been suggested by G. Walker and colleagues34 who proposed DNA replication that the RecA-mediated conversion of uncleaved UmuD to UmuDЈ is a key regu- Persisting latory event in the resumption of DNA lesions synthesis following exposure to UV light. It would seem that the delay in the oc- (c) Pol V Mut currence of these two processes plays RecA* an important role in separating error- 3′ T T free and error-prone mechanisms of G A replication restart. The observations that recFR mutants were also delayed in the resumption of DNA synthesis and Pol III preferentially degraded growing fork Pol V DNA after UV exposure (Ref. 35) suggest that RecFR can help to protect the Pol V nascent strands of a disrupted repli- cation fork. A recent review discusses the possible involvement of RecFOR in 3′ T T catalysing RecA filament formation at G A UV-generated daughter-strand gaps36. In summary, we suggest that a process- Pol III HE ive Pol II HE (i.e. involving the ␤,␥ com- Ti BS plex37, acting in concert with a restart primosome composed of PriA, PriB, Figure 3 PriC, DnaT, DnaB, DnaC and DnaG pro- Temporal steps governing the catalysis of DNA polymerase II (Pol II)-catalysed error-free 38 replication-restart and Pol V-catalysed error-prone translesion synthesis. (a) Pol III holoenzyme teins ) are instrumental in catalysing (HE) encounters a DNA template lesion. Stalling and disassembly of replicative Pol III HE at rapid replication-restart repair synthesis a template lesion site, shown as a 6-4 T–T photoproduct, accompanied by a brief inhibition beginning almost immediately after UV of DNA synthesis, is followed within a matter of minutes by (b) error-free Pol II-dependent irradiation in an error-free UV repair replication-restart synthesis involving PriA as part of a restart primosome38. The restart pathway (Fig. 3). In contrast, Pol V cataly- primosome is composed of PriA, PriB, PriC, DnaT, DnaB, DnaC and DnaG proteins38. Acting ses error-prone translesion synthesis, as a specificity element, PriA directs the loading of DnaB and DnaG proteins at a D-loop on an invading DNA strand38, enabling synthesis of a short RNA primer, elongated subsequently coming into play almost an hour later by E. coli Pol II. Reassembly of a replication fork containing Pol III HE takes place shortly (Fig. 3). Therefore, Pol II and Pol V appear thereafter. (c) Approximately 50 min later, Pol V Mut-catalysed error-prone translesion synthesis to act in the cell as ‘flip sides of a coin’. occurs at persisting template lesions, as shown in Fig. 2. Here we show Pol V Mut incorporating G and A opposite the 3Ј- and 5Ј-sites of a 6-4 T–T photoproduct, respectively, in accordance Role of replicative Pol III in SOS repair with in vitro and in vivo data15. In contrast to error-prone translesion synthesis, Pol II-catalysed What is the role of Pol III HE in the replication restart is an error-free process that is likely to involve RecA-dependent D-loop Pol II error-free and Pol V error-prone formation, strand invasion and branch migration reactions, followed by Pol III-dependent DNA synthesis and subsequent lesion repair. Other proteins implicated in replication repair pathways? Replication restart restart include RecFO and RecFR, which could be involved either directly or perhaps indi- experiments performed in a temperature- rectly by aiding in RecA* filament formation. Although the biochemical steps in replication sensitive Pol III background showed that restart remain to be elucidated, the proteins involved have been identified genetically. following the initiation of replication Daughter DNA strands are shown in red, the invading D-loop strand is shown in blue. 193 REVIEWS TIBS 25 – APRIL 2000

site or opposite G, Rev1p is serving as a I II III IV V template-dependent dCMP transferase47, N C Ec UmuC (422) not as a template-directed DNA polym- DinB/Rad30 erase. Therefore, unlike Pol V, Rev1p does not effectively catalyse all four N C h RAD30B/polι (715) DinB canonical Watson–Crick base pairs at natural, undamaged DNA template sites N C C2HC Ec DINB/polIV (351) ? ? (it incorporates C opposite both G and N C h DINB1 (870) abasic sites), whereas Pol V strongly fa- vors synthesis of canonical A–T and G–C N Ce F22B7.6 (598) C base pairs on undamaged DNA, albeit C H Rad30 2 2 with extremely low fidelity (~10Ϫ3–10Ϫ4)25. η N C h RAD30A/pol (713) Although numerous proteins are in- N C Sc Rad30p (632) duced in response to DNA damage in all

BRCT organisms studied to date, it remains N C Sc Rev1p (985) uncertain whether eukaryotic cells con- tain analogous tightly regulated SOS N C h REV1 (1251) functions, especially as apoptosis pro- Ti BS vides an alternative route for cells with heavily damaged DNA. However, one Figure 4 can easily imagine how promiscuous UmuC/DinB/Rev1/Rad30 DNA polymerase superfamily alignment. Highly conserved polymerases (i.e. enzymes with lowered domains I–V containing putative catalytic residues and helix-hairpin-helix DNA-binding motifs fidelity) might be used to generate are denoted by roman numbers. UmuC is least conserved followed by human RAD30B, which shares an extra region of homology found in the DinB and Rad30 subgroups (light-blue rect- hypermutation in the V(D)J coding region angles). UmuC and Rad30B have unique C-terminal ends (thin black lines). The DinB subgroup in human B cells to aid in generating di- exhibits three conserved short motifs (violet rectangles) present from E. coli to humans. verse groups of antibodies48, while also

Zinc-binding motifs C2H2 and C2HC (dark-gray and yellow diamonds, respectively) are thought providing organisms with the potential to be involved in DNA binding and selective targeting. The BRCT domain (brown) is shown at the for adaptation and evolution. N-terminal end of the Rev1 subgroup. Conserved regions of unknown function are found in the N-terminal region (purple ovals) and C-terminal region (red squares) of human and C. elegans Acknowledgements DinB. Additional motifs conserved within subgroups are shown in gray. Amino acid lengths I want to acknowledge the truly re- are shown in parentheses. Ec (E. coli), h (human), Ce (C. elegans), Sc (S. cerevisiae). markable contributions made by Evelyn Witkin, Miroslav Radman and Graham restart by Pol II and PriA, Pol III takes UmuC/DinB/Rev1/Rad30 superfamily of Walker in the intellectual and experi- over from Pol II to continue chromo- errant DNA polymerases is represented mental development of the field of SOS somal replication6. It seems likely that a in all prokaryotic and eukaryotic organ- mutagenesis and repair. I owe a special fully bidirectional replication complex isms investigated to date (reviewed in debt of gratitude to Hatch Echols for his involving Pol III HE is reformed at this Refs 39–41). Figure 4 contains a sequence pioneering work in development of the point in time. We suggest that the error- alignment for several of its members. first biochemical reconstitution assay free Pol II replication restart pathway is The sizable number of protein com- for translesion synthesis and for his likely to involve homologous recombi- ponents required for Pol V Mut-catalysed cherished friendship. I also wish to nation with the direct involvement of translesion synthesis – RecA, the ␤,␥ thank my colleagues Roger Woodgate, RecA in the formation of D-loops, which complex and SSB – provides a unique Mike O’Donnell, Ramon Eritja, Kevin are then used by a restart primosome38 biochemical signature that distinguishes McEntee, and John-Stephen Taylor and, and Pol II to initiate replication restart. Pol V from the other prokaryotic and of course, my past and present students Elucidation of the biochemical reactions eukaryotic polymerase homologs. E. coli for their innumerable contributions to involved in replication restart must await Pol IV (DinB), for example, appears to all aspects of this work. This work was the successful reconstitution of an in vitro extend mismatched base pairs efficiently supported by NIH grants GM42554 and assay for this process. A model illustrat- in the absence of other proteins42. In the GM21422. ing a likely sequence of events taking case of yeast Pol ␩ (Rad30p)43, and pre- place during error-free replication restart sumably its human XPV homolog44,45, References 1 Walker, G.C. (1985) Inducible DNA repair systems. and error-prone translesion synthesis is cis-syn T–T photodimers can be copied Annu. Rev. Biochem. 54, 425–457 shown in Fig. 3. Pol III HE is thought to accurately by incorporating consecutive 2 Bruck, I. et al. (1996) Purification of a soluble UmuDЈC complex from Escherichia coli: cooperative play a similar role in both replication A’s, without the intervention of auxiliary binding of UmuDЈC to single-stranded DNA. J. Biol. restart and translesion synthesis path- proteins. However, Pol ␩ is highly error- Chem. 271, 10767–10774 46 3 Tang, M. et al. (1998) Biochemical basis of ways by replacing Pol II and Pol V to prone when copying natural DNA . In SOS mutagenesis in Escherichia coli: reconstitution complete chromosomal replication. contrast, Pol V is prone to make errors of in vitro lesion bypass dependent on the Ј UmuD2 C mutagenic complex and RecA protein. both when replicating a variety of lesions Proc. Natl. Acad. Sci. U. S. A. 95, 9755–9760 Ј Eukaryotic homologs of UmuC/DinB-like and when copying undamaged DNA tem- 4 Tang, M. et al. (1999) UmuD 2C is an error-prone polymerases plate sites3,25,31. An important distinction DNA polymerase, Escherichia coli pol V. Proc. Natl. Acad. Sci. U. S. A. 96, 8919–8924 Replication train wrecks are not con- between Pol V and yeast Pol ␨ (comprised 5 Reuven, N.B. et al. (1999) The mutagenic fined to prokaryotic cells, and neither of the Rev3 and Rev7 proteins) is that protein UmuC is a DNA polymerase activated by ␨ UmuDЈ, RecA and SSB and is specialized for are the polymerases required to copy when Pol interacts with Rev1p and in- translesion replication. J. Biol. Chem. 274, damaged DNA. A recently discovered corporates C opposite an abasic template 31763–31766 194 TIBS 25 – APRIL 2000 REVIEWS

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Over the next few months TiBS will publish articles that review different aspects of mitochondrial function. Articles will include: Regulation of mitochondrial metabolism by ER Ca2؉ release: an intimate connection Guy Rutter and Rosario Rizzuto Insights into the internal structure of the mitochondria Terry Frey and Carmen Mannella Bioenergetics Vladimir Skulachev Mitochondria, free radicals and aging Brian Robinson Mitochondrial genetics and disease Eric Schon The role of mitochondria in cell death Paolo Bernardi

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