Differential correction of lagging-strand replication errors made by DNA polymerases α and δ

Abstract

Mismatch repair (MMR) of replication errors requires DNA ends that can direct repair to the newly synthesized strand containing the error. For all but those organisms that use adenine methylation to generate nicks, the source of these ends in vivo is unknown. One possibility is that MMR may have a "special relation to the replication complex" [Wagner R, Jr., Meselson M (1976) Proc Natl Acad Sci USA 73: [4135][4136][4137][4138][4139], perhaps one that allows 5′ or 3′ DNA ends associated with replication to act as strand discrimination signals. Here we examine this hypothesis, based on the logic that errors made by yeast DNA polymerase α (Pol α), which initiates Okazaki fragments during lagging-strand replication, will always be closer to a 5′ end than will be more internal errors generated by DNA polymerase δ (Pol δ), which takes over for Pol α to complete lagging-strand replication. When we compared MMR efficiency for errors made by variant forms of these two polymerases, Msh2-dependent repair efficiencies for mismatches made by Pol α were consistently higher than for those same mismatches when made by Pol δ. Thus, one special relationship between MMR and replication is that MMR is more efficient for the least accurate of the major replicative polymerases, exonuclease-deficient Pol α. This observation is consistent with the close proximity and possible use of 5′ ends of Okazaki fragments for strand discrimination, which could increase the probability of Msh2-dependent MMR by 5′ excision, by a Msh2-dependent strand displacement mechanism, or both.replication fork | DNA repair | infidelity | mutagenesis | base substitutions V arious types of DNA repair, including base excision repair, nucleotide excision repair, double-strand DNA break repair, and mismatch repair (MMR), are differentiated into subpathways that can be distinguished by substrate specificity and enzymology (1). This is particularly true for DNA mismatch repair (reviewed in refs. 2-6), which can involve any of several different protein complexes at sequential steps in the pathway. For example, repair of mismatches generated during eukaryotic DNA replication is initiated upon mismatch binding by Msh2-Msh6 or Msh2-Msh3, and subsequent steps can involve three different MutL heterodimers (reviewed in ref. 5). Starting from a nick in the nascent DNA strand, mismatches can be removed by excision. Recent evidence suggests that mismatches may also sometimes be removed by a strand displacement mechanism (7). In either case, removal of the mismatch requires a DNA strand discontinuity. Studies in vitro demonstrate that this discontinuity can be a nick located either 3′ or 5′ to the mismatch, with the protein requirements for MMR differing somewhat depending on the location of the nick relative to the mismatch (see refs. 2 and 7 and references therein).Early experiments in Escherichia coli revealed that one signal that directs MMR to the nascent strand is transient under-methylation of adenine in GATC sequences, allowing MutH endonuclease to nick that strand to d...

Postreplicative Mismatch Repair

“…Evidence in support of this hypothesis came from in vivo studies in S. cerevisiae showing that the lagging strand was more efficiently repaired by MMR (Pavlov et al 2003;Nick McElhinny et al 2010) possibly due to the ready availability of DNA termini where MMR could initiate. The hypothesis could also be substantiated by in vitro studies using extracts of human cells and circular substrates carrying single mismatches.…”

Replicative DNA Polymerases

“…Thus, the absence of a proofreading activity in Pol a requires that errors made by it are corrected by other mechanisms. For instance Pol d, but not Pol 1, proofreads errors made by Pol a, in addition to error correction by the mismatch repair system (Pavlov et al 2006;Nick McElhinny et al 2010a).…”

An end for mismatch repair

“…One of the first papers to describe MMR suggested that the MMR repair complex could have a special relationship with the replication apparatus (4), and that idea seems to be a central feature of strand discrimination in eukaryotes. In a study published in PNAS, Nick McElhinny et al (5) provide evidence that the close relationship between MMR and replication leads to preferential repair of mismatches near initiation sites for DNA synthesis.…”

“…Determination of MMR correction with regard to Pol ε errors and individual measurements of MutSα and MutSβ correction activity, all of which are underway, should be especially revealing. The recent papers in PNAS (5,11) show the relationship of MMR with the replication apparatus to be critical in its strand discrimination activity. The paper by Pluciennik et al (11) is particularly important in illustrating how strand discrimination can be achieved on the leading strand, and Nick McElhinny et al (5) demonstrate that the 5′ ends of Okazaki fragments do, in fact, appear to be used for strand discrimination on the lagging strand.…”

“…The recent papers in PNAS (5,11) show the relationship of MMR with the replication apparatus to be critical in its strand discrimination activity. The paper by Pluciennik et al (11) is particularly important in illustrating how strand discrimination can be achieved on the leading strand, and Nick McElhinny et al (5) demonstrate that the 5′ ends of Okazaki fragments do, in fact, appear to be used for strand discrimination on the lagging strand. Although we are now much closer to understanding how eukaryotic MMR achieves strand discrimination when correcting replication errors, there remain additional complexities to be explored.…”