Response to Burgers et al.
2016; Elsevier BV; Volume: 61; Issue: 4 Linguagem: Inglês
10.1016/j.molcel.2016.01.018
ISSN1097-4164
AutoresRobert E. Johnson, Roland Klassen, Louise Prakash, Satya Prakash,
Tópico(s)Fungal and yeast genetics research
ResumoIn our study (Johnson et al., 2015Johnson R.E. Klassen R. Prakash L. Prakash S. Mol. Cell. 2015; 59: 163-175Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), we concluded that DNA polymerase (Pol) δ replicates both the leading and lagging DNA strands and that Polε plays no significant role in leading-strand replication. In their Letter in this issue of Molecular Cell, Burgers et al., 2016Burgers P.M.J. Gordenin D.A. Kunkel T.A. Mol. Cell. 2016; 61 (this issue): 492-493Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar contend that their model wherein Polε primarily replicates the leading strand still remains valid and suggest that (1) our strains contain suppressors, (2) our observed G→T mutations originate in the lagging strand, and (3) ribonucleotide incorporation data support their model. The Kunkel group analyzed mutation rates in haploid spores derived from their yeast strain Δl(−2)l-7B-YUNI300 because their pol3-L612M msh2Δ strain exhibits growth defects and heterogeneous colony size and because such growth defects can give rise to suppressor mutations. However, our pol3-L612M msh2Δ strains displayed no growth defects in either the S288C or the DBY747 background, nor in the pol3-L612M msh2Δ double mutants obtained by tetrad analysis of POL3/pol3-L612M MSH2/msh2Δ diploids (Figure S5 in Johnson et al., 2015Johnson R.E. Klassen R. Prakash L. Prakash S. Mol. Cell. 2015; 59: 163-175Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). We show that in the pol3-L612M S288C strain, Polδ signature errors are detectable only in the lagging strand, whereas in the pol3-L612M msh2Δ strain, they also accumulate on the leading strand. Since in the pol3-L612M msh2Δ strain ∼80% of all mutations are L612M-Polδ signature mutations, a large majority of mutations in this strain represent Polδ-generated errors during replication. Moreover, since suppression could explain the reduced but not the highly increased leading-strand signature mutations of L612M-Polδ that we observe in the pol3-L612M msh2Δ strain (see below), suppression is not affecting any of our conclusions for Polδ's role in replication. Kunkel and colleagues state that since the G→T hotspots at base pairs 679 and 706 are missing in our strain having URA3 in the opposite orientation, this would lead to the paradoxical suggestion that L612M-Polδ does not replicate the lagging strand. However, such orientation dependence of hotspot errors can be seen in the study by Kunkel and colleagues; although there is a strong T→C hotspot at position 97 in OR1 in URA3, this hotspot is missing in URA3 in the opposite orientation, and no complementary A→G mutations were observed (Nick McElhinny et al., 2008Nick McElhinny S.A. Gordenin D.A. Stith C.M. Burgers P.M.J. Kunkel T.A. Mol. Cell. 2008; 30: 137-144Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). In our paper, we stress the point that although the L612M-Polδ-generated hotspot mutations occur on both the DNA strands in the pol3-L612M msh2Δ S288C strain, the sites at which hotspot mutations occur differ in an orientation-dependent manner, and mismatch repair (MMR) and L612M-Polδ mispair generation can act differentially at different sites during replication of the two DNA strands. Furthermore, differential contribution of MMR and other mismatch removal processes can account for the variability in the level of increase in mutation rates in different yeast strains (Johnson et al., 2015Johnson R.E. Klassen R. Prakash L. Prakash S. Mol. Cell. 2015; 59: 163-175Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Even though we observe a large increase in the rate of G→T hotspot mutations at base pairs 679 and 706 in URA3, which we attribute to errors made by Polδ during leading strand replication (see Figure 2B in Johnson et al., 2015Johnson R.E. Klassen R. Prakash L. Prakash S. Mol. Cell. 2015; 59: 163-175Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), Kunkel and colleagues contend that these mutations arise from C:dTTP mispair formation on the lagging strand; but they provide no rationale for this. In their biochemical studies, they showed that L612M-Polδ exhibits an 8.5:1 bias for G:dATP mispair formation over the C:dTTP mispair (Nick McElhinny et al., 2007Nick McElhinny S.A. Stith C.M. Burgers P.M. Kunkel T.A. J. Biol. Chem. 2007; 282: 2324-2332Crossref PubMed Scopus (66) Google Scholar), and we independently confirmed this in the sequence context of position 679 in URA3 (see Figures S2C and S2D in Johnson et al., 2015Johnson R.E. Klassen R. Prakash L. Prakash S. Mol. Cell. 2015; 59: 163-175Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Therefore, their claim that the observed G→T mutations derive from C:dTTP mispairs on the lagging strand is not supported by experimental evidence. In their recent study which analyzed the whole-genome sequence of a pol3-L612M msh2Δ homozygous diploid, they identified strand-specific G→T mutations near origins. The G:dATP bias predicts that these mutations arise primarily from G:dATP mispair formation on the leading strand, and not from C:dTTP mispair formation on the lagging strand, as they suggest (Lujan et al., 2014Lujan S.A. Clausen A.R. Clark A.B. MacAlpine H.K. MacAlpine D.M. Malc E.P. Mieczkowski P.A. Burkholder A.B. Fargo D.C. Gordenin D.A. Kunkel T.A. Genome Res. 2014; 24: 1751-1764Crossref PubMed Scopus (104) Google Scholar). Thus, even in their strain, there is evidence for leading-strand replication by Polδ. We provide extensive evidence for the various L612M-Polδ signature mutations on both DNA strands in the pol3-L612M msh2Δ S288C strain and also in the pol3-L612M msh2Δ DBY747 strain in which URA3 was integrated at many different genomic locations. Altogether, our data support the conclusion that Polδ replicates both the DNA strands. Furthermore, since Polε signature errors on the leading strand do not occur in the pol2-M644G msh2Δ strain, Polε plays little if any role in leading-strand replication (Johnson et al., 2015Johnson R.E. Klassen R. Prakash L. Prakash S. Mol. Cell. 2015; 59: 163-175Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). In view of our data (Johnson et al., 2015Johnson R.E. Klassen R. Prakash L. Prakash S. Mol. Cell. 2015; 59: 163-175Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), we consider it highly likely that explanations other than a role of Polε in the replication of the leading strand account for the increase in ribonucleotides on the nascent leading strand in the RNase H2-deficient pol2-M644G mutant. We suggested in our paper that in yeast strains harboring the pol2-M644G mutation, because of the greatly enhanced capacity of the M644G-Polε over wild-type Polδ (∼50-fold) to incorporate ribonucleotides and to extend synthesis from them (Lujan et al., 2013Lujan S.A. Williams J.S. Clausen A.R. Clark A.B. Kunkel T.A. Mol. Cell. 2013; 50: 437-443Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), the mutant Polε takes over synthesis from Polδ and promotes the persistence of ribonucleotides incorporated by Polδ on the leading strand. Moreover, since checkpoint pathways are activated and dNTP levels are elevated in pol2-M644G cells (Williams et al., 2015Williams L.N. Marjavaara L. Knowels G.M. Schultz E.M. Fox E.J. Chabes A. Herr A.J. Proc. Natl. Acad. Sci. USA. 2015; 112: E2457-E2466Crossref PubMed Scopus (40) Google Scholar), these factors would contribute to a further increase in the proficiency of mutant Polε to extend synthesis from ribonucleotides. As for the observation that in the pol3-L612M rnh201Δ strain enhanced ribonucleotide incorporation is detected on the lagging strand, we suggest that even though Polδ incorporates ribonucleotides on both the DNA strands, they are more efficiently removed from the leading strand by competing pathways. The identity of these pathways remains to be determined, but because Polε exonuclease can excise ribonucleotides (Williams et al., 2012Williams J.S. Clausen A.R. Nick McElhinny S.A. Watts B.E. Johansson E. Kunkel T.A. DNA Repair (Amst.). 2012; 11: 649-656Crossref PubMed Scopus (52) Google Scholar), this proofreading exonuclease may also act in one such competing pathway. Contrary to their statement that Polε does not proofread mistakes made by Polδ, we provide evidence for Polε exonuclease in removing Polδ errors (see Table 1 in Johnson et al., 2015Johnson R.E. Klassen R. Prakash L. Prakash S. Mol. Cell. 2015; 59: 163-175Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Burgers et al., 2016Burgers P.M.J. Gordenin D.A. Kunkel T.A. Mol. Cell. 2016; 61 (this issue): 492-493Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar support their statement by citing a recent study Flood et al., 2015Flood C.L. Rodriguez G.P. Bao G. Shockley A.H. Kow Y.W. Crouse G.F. PLoS Genet. 2015; 11: e1005049Crossref PubMed Scopus (41) Google Scholar, which is based on an a priori assumption that Polδ replicates the lagging strand and that Polε replicates the leading strand, and it was not designed to directly test the role of Polε exonuclease in removing Polδ-generated mispairs from the leading strand, as we have done. Without a more complete understanding of the roles of Polδ and Polε in ribonucleotide incorporation and the roles of ribonucleotide removal pathways on each DNA strand, it seems inappropriate to dismiss the evidence indicating a role of Polδ, but not of Polε, in replicating the leading strand, and to selectively use the ribonucleotide incorporation data to propose a role for Polε in replicating the leading strand. While in vitro studies support an ability of Polε to carry out DNA synthesis on the leading strand (Georgescu et al., 2014Georgescu R.E. Langston L. Yao N.Y. Yurieva O. Zhang D. Finkelstein J. Agarwal T. O'Donnell M.E. Nat. Struct. Mol. Biol. 2014; 21: 664-670Crossref PubMed Scopus (141) Google Scholar), it remains possible that such studies fail to recapitulate all the molecular complexities that occur during replication. Polε plays an essential role in the assembly and progression of CMG helicase on the leading strand, but its polymerase function is dispensable for viability. We have suggested a role for its polymerase activity in rescuing the replication fork at sites where Polδ stalls on the leading strand and in other DNA repair processes, and for its proofreading activity in the removal of Polδ-generated errors (Johnson et al., 2015Johnson R.E. Klassen R. Prakash L. Prakash S. Mol. Cell. 2015; 59: 163-175Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). The elucidation of these and other Polε roles would require a thorough genetic and molecular analysis of the complexities that underlie these Polε functions. R.E.J. and R.K. analyzed data. R.E.J., R.K., L.P., and S.P. interpreted results and wrote the letter. A Major Role of DNA Polymerase δ in Replication of Both the Leading and Lagging DNA StrandsJohnson et al.Molecular CellJuly 2, 2015In BriefThe current model of eukaryotic DNA replication suggests that DNA polymerase (Pol)ε primarily replicates the leading strand while Polδ replicates the lagging strand. Johnson et al. provide genetic evidence that Polδ replicates both strands, while Polε's proofreading activity is important for removing Polδ-generated errors from the leading strand. Full-Text PDF Open ArchiveWho Is Leading the Replication Fork, Pol ε or Pol δ?Burgers et al.Molecular CellFebruary 18, 2016In BriefSeveral studies in the past decade support a model wherein DNA polymerase ε (Pol ε) carries out the majority of leading-strand DNA replication of the undamaged eukaryotic nuclear genome. Now a recent paper in Molecular Cell from the Prakash laboratory challenges this model, claiming instead that Pol δ is the major replicase for both strands and that Pol ε's primary role is only to proofread errors made by Pol δ during leading-strand replication (Johnson et al., 2015). While we fully subscribe to the idea that the replication fork is plastic and that its composition can adapt to various challenges, we believe the foundation for an unchallenged replication fork remains as established before the Prakash paper, for the following reasons. Full-Text PDF Open Archive
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