A Major Role of DNA Polymerase δ in Replication of Both the Leading and Lagging DNA Strands

Abstract

Genetic studies with S. cerevisiae Polδ (pol3-L612M) and Polε (pol2-M644G) mutant alleles, each of which display a higher rate for the generation of a specific mismatch, have led to the conclusion that Polε is the primary leading strand replicase and that Polδ is restricted to replicating the lagging strand template. Contrary to this widely accepted view, here we show that Polδ plays a major role in the replication of both DNA strands, and that the paucity of pol3-L612M-generated errors on the leading strand results from their more proficient removal. Thus, the apparent lack of Polδ contribution to leading strand replication is due to differential mismatch removal rather than differential mismatch generation. Altogether, our genetic studies with Pol3 and Pol2 mutator alleles support the conclusion that Polδ, and not Polε, is the major DNA polymerase for carrying out both leading and lagging DNA synthesis.

Figure 1. Orientation bias of ura3-104 reversion in the S288C pol3-L612M strains defective in Polε proofreading (pol2-4), MMR (msh2Δ) or Exo1 (exo1Δ

(A) CAG-specific reversion rates of ura3-104 for various strains in orientations OR1 and OR2. (B) Fold increase in CAG-specific reversion rates in either OR1 or OR2. (C) Reversal of strand bias in Pol3-L612M mutation generation from lagging to leading strand by inactivation of mismatch removal processes. See also Figures S1-S5.

Figure 2. Lack of strand bias of URA3 mutations near ARS306 in the S288C pol3-L612M msh2Δ strain

(A) Mispair generation bias of L612M Polδ. Point mutations are shown above the two mispairs that generate the mutation. The bias of L612M Polδ for each mispair is given below (Nick McElhinney, 2007) (B) Hot spot mutations in URA3 observed in OR1 in the S288C pol3-L612M msh2Δ strain. The orientation of the URA3 ORF (boxed arrow) is depicted by the direction of the arrow. URA3, integrated ~1.2 kb to the left of ARS306 in chromosome 3 is shown schematically and is not drawn to scale. Each hot spot is shown by their respective base pairs, and their positions in the URA3 ORF are shown within the boxed arrow. Base changes generated during the replication of the leading strand (above) and the lagging strand (below) are shown. Filled in triangles represent −1 frameshift mutations. The proportion of observed mutations at each site were assigned to the lagging and leading strand according to the bias for mispair formation by L612M Polδ shown in (A). Strand specific mutation rates for each site were calculated as described in the text, and the leading and lagging strand hot spot mutation rates given on the far right are the sum of all hot spot mutations on that strand. (C) Hot spot mutations in URA3 observed in OR2 in the pol3-L612M msh2Δ strain. The orientation of URA3 is reversed from that in (B). See also Figures S2, S4, S5, and Tables S1 and S2.

Figure 3. Lack of strand bias of URA3 mutations located near ARS306 in the DBY747 pol3-L612M msh2Δ strain

(A) Hot spot mutations in URA3 observed in OR1 in the DBY747 pol3-L612M msh2Δ strain. The URA3 coding region is integrated ~ 1.2 kb to the left of ARS306 on chromosome 3 as described in Figure 2. (B) Hot spot mutations in URA3 near ARS306 observed in OR2 in the pol3-L612M msh2Δ strain. The orientation of URA3 is reversed from that in (A). The sum of all hotspot mutations for the leading or the lagging DNA strand is given on the right. See also Figure S4 and Table S3.

Figure 4. Lack of strand bias of URA3 mutations located ~10kb left of ARS306 in the DBY747 pol3-L612M msh2Δ strain

(A) Hot spot mutations in URA3 observed in OR1 in the DBY747 pol3-L612M msh2Δ strain. The URA3 ORF (boxed arrow) is integrated ~10 kb to the left of ARS306 on chromosome 3 in the intergenic region between the STE50 and RRP1 genes. (B) Hot spot mutations observed in URA3 located ~10 kb to the left of ARS306 in OR2 in the DBY747 pol3-L612M msh2Δ strain. The orientation of URA3 is reversed from that in (A). The sum of all hotspot mutations for the leading or the lagging DNA strand is given on the right. See also Table S4.

Figure 5. Lack of strand bias of URA3 mutations in the DBY747 pol3-L612M msh2Δ strain at ARS1

(A) Hot spot mutations in URA3 observed in OR1 in the DBY747 pol3-L612M msh2Δ strain. The URA3 ORF (boxed arrow) is located ~600 bp to the left of ARS1 on chromosome 4 and is not drawn to scale. Strand specific mutation rates for each site were calculated as described in the text, and the leading and lagging strand hot spot mutation rates given on the far right are the sum of all hot spot mutations on that strand. (B) Hot spot mutations in URA3 near ARS1 observed in OR2 in the DBY747 pol3-L612M msh2Δ strain. The orientation of URA3 is reversed from that in (A). See also Figure S7 and Tables S5 and S6.

Figure 6. Lack of pol2-M644G signature errors in MMR deficient strains

(A) Mutational bias for A to T and T to A mutations in pol2-M644G and pol2-M644G msh2Δ strains. The T:A to A:T mutation is shown above the 2 mispairs that cause the mutation. The signature bias exhibited by pol2-M644G is indicated (Pursell et al., 2007). The number of spontaneous A to T and T to A mutation events as well as hotspot mutations A279T and A686T in pol2-M644G and pol2-M644G msh2Δ strains harboring URA3 in orientation 1 (OR1) or in orientation 2 (OR2) are shown. The location of the T:dTTP mispair in the leading (lead) or lagging (lag) strand in each orientation is shown and the ratio of leading strand versus lagging strand mutations is given. (B) and (C) Schematic representation of the leading and lagging strand specific mutational events at A279 and A686 in URA3 in OR1 or in OR2 in the pol2-M644G (B) and pol2-M644G msh2Δ (C) strain.