Quantifying the contributions of base selectivity, proofreading and mismatch repair to nuclear DNA replication in Saccharomyces cerevisiae

Abstract

Mismatches generated during eukaryotic nuclear DNA replication are removed by two evolutionarily conserved error correction mechanisms acting in series, proofreading and mismatch repair (MMR). Defects in both processes are associated with increased susceptibility to cancer. To better understand these processes, we have quantified base selectivity, proofreading and MMR during nuclear DNA replication in Saccharomyces cerevisiae. In the absence of proofreading and MMR, the primary leading and lagging strand replicases, polymerase ε and polymerase δ respectively, synthesize DNA in vivo with somewhat different error rates and specificity, and with apparent base selectivity that is more than 100 times higher than measured in vitro. Moreover, leading and lagging strand replication fidelity rely on a different balance between proofreading and MMR. On average, proofreading contributes more to replication fidelity than does MMR, but their relative contributions vary from nearly all proofreading of some mismatches to mostly MMR of other mismatches. Thus accurate replication of the two DNA strands results from a non-uniform and variable balance between error prevention, proofreading and MMR.

Eukaryotic genome instability in light of asymmetric DNA replication

“…In the absence of proofreading and MMR of base substitutions ( msh6Δ ), studies of replication error specificity in vivo (St Charles et al, 2015) suggest that exonuclease-deficient yeast Pols δ and ε have apparent nucleotide selectivities of >10 6 and >10 7 , respectively. That is, an incorrect dNTP is selected less than once per million bases synthesized by Pol δ and per ten million bases synthesized by Pol ε.…”

“…Thus both polymerases are highly accurate. A comparison of these rates to the rates in cells that are deficient in only MMR (St Charles et al, 2015) indicates that base-base mismatches are also efficiently proofread by Pols δ and ε. Many of these mismatches are likely to be proofread intrinsically by the exonuclease activity of the polymerase that made the mismatch (Figure 2, panels C and E).…”

“…In fact, mismatches made by variants of Pol ε are often repaired by Msh2-dependent MMR as efficiently, and often more efficiently, than mismatches made by variants of Pols α and δ (Lujan et al, 2014, Lujan et al, 2012, St Charles et al, 2015). Thus, in addition to Exo1 digestion from the 5′ DNA ends associated with Okazaki fragments, other means of mismatch removal are available.…”

Human mismatch repair system balances mutation rates between strands by removing more mismatches from the lagging strand

“…Its fidelity is achieved through three main components: base selectivity of polymerases, proofreading activity of their exonuclease domains, and repair of mismatches that escaped proofreading by the mismatch repair (MMR) system (Kunkel 2009). Studies in yeast indicate that the effectiveness of each of these steps depends on the mismatch type and that MMR compensates for the infidelity of polymerases (Kunkel 2011;St Charles et al 2015). The classical model of the eukaryotic replication fork (Larrea et al 2010) suggests a division of labor in replication of the leading and lagging strands among the major DNA polymerases, with polymerase epsilon (Pol epsilon) replicating the leading strand and polymerases alpha (Pol alpha) and delta (Pol delta) replicating the lagging strand, with the possible exception of replication origins and other specific regions where Pol delta may contribute to replication of both strands (Yeeles et al 2017).…”

“…Under this model, the asymmetry in mutation rates between the leading and the lagging DNA strands may arise due to differences in fidelity of polymerases replicating these strands. In yeasts, different replicative polymerases possess different biases in the types of mutations they introduce, leading to differences in mismatch types between the leading and the lagging strands (St Charles et al 2015).…”