Dividing the workload at a eukaryotic replication fork

Abstract

Efficient and accurate replication of the eukaryotic nuclear genome requires DNA polymerases (Pols) alpha, delta and epsilon. In all current replication fork models, polymerase alpha initiates replication. However, several models have been proposed for the roles of Pol delta and Pol epsilon in subsequent chain elongation and the division of labor between these two polymerases is still unclear. Here, we revisit this issue, considering recent studies with diagnostic mutator polymerases that support a model wherein Pol epsilon is primarily responsible for copying the leading-strand template and Pol delta is primarily responsible for copying the lagging-strand template. We also review earlier studies in light of this model and then consider prospects for future investigations of possible variations on this simple division of labor.

Figure 1

Assigning yeast Pol δ and Pol ε to specific strands. (a) During DNA synthesis in vitro, L612 M Pol δ generates T-dGMP errors at a rate that is at least 28-fold that of A-dCMP errors (green). (b) In a pol3-L612 M msh2^- yeast strain, the T-A to C-G mutation rate (depicted earlier as the T to C substitution and depicted here as the inferred T-G mismatch [23]) is high (58 × 10^-7) at base pair 97 in URA3 when present in orientation 1. Given the biased error rates in panel (a), these mutations are inferred to result from T-dGMP errors during lagging-strand synthesis by Pol δ. (c) In orientation 2, the mutation rate for the same base pair (base pair 97) in the same neighboring sequence context is much lower (3.10^-7), implying that Pol δ has little role in leading-strand synthesis. Using this same logic, because Pol δ deletes template T in homopolymeric runs at a rate 11-fold that of what it deletes in template A [see (a)], L612 M Pol δ is inferred to delete a T-A base pair from a run of five T-A base pairs (174-178) during lagging-strand replication of template Ts [see (c)], but not during leading-strand replication of template As [see (b)]. Finally, note that M644G Pol ε generates T-dTMP errors at a rate ≥39-fold that of A-dAMP errors [blue in part (a)]. In a pol2-M644G strain, the T-A to A-T mutation rate is higher at base pair 686 in URA3 orientation 1. Given the biased error rates in panel (a), these mutations are inferred to result from T-dTMP errors during leading-strand replication by M644G Pol ε. In orientation 2 [see (c)], the mutation rate for the same base pair (base pair 686) in the same neighboring sequence context is much lower, implying that Pol ε has little role in lagging-strand synthesis. Additional examples of mutational specificity that are consistent with these interpretations can be found in Refs [12,23]. Part (a) adapted from Refs. [12,22]. Parts (b,c) adapted from Refs [12,23].

Figure 2

Models for eukaryotic DNA replication forks. On the left is a model illustrating primary roles for Pol ε and Pol δ in leading- and lagging-strand replication, respectively. Other proteins shown include the Pol α-primase (red), the MCM helicase (yellow), the eukaryotic single-stranded-DNA-binding protein, replication protein A (RPA; gray), the sliding clamp proliferating cell nuclear antigen (PCNA; green) and the FEN1-DNA ligase complex (yellow-red). On the right is a model wherein Pol ε dysfunction causes formation of an alternative fork. Conditions other than Pol ε dysfunction might also cause formation of alternative forks and such forks could be assembled at origins, by remodeling the normal fork or during replication restart after an encounter with a natural replication barrier or a lesion.

Figure 3

Fork integrity maintained over a 34 kb replicon. (a) Map of the left arm of yeast chromosome III. Distances between elements are in thousands of base pairs and are shown above the chromosome. The region examined is expanded below the chromosome with locations and names of insertion alleles. Replication origins are shown as black rectangles. (b) Ratio of reversion rate of the ura3-29 allele in orientation 1 versus orientation 2 at different locations in chromosome III in an ogg1 strain that is defective in OGG1, the DNA glycosylase that removes the pre-mutagenic 8-oxoG lesion from DNA. Black rectangles indicate two functional origins. Each bar represents the reversion rate ratio at the location corresponding to the position in panel (a). The scheme below each of the bar graphs depicts the region of chromosome III undergoing bidirectional replication initiated at ARS305 and the ARS306. Continuous arrows are for leading-strand replication and multiple arrows represent lagging-strand replication. Replication forks move to the left and to the right from each origin and meet at a site that is equidistant from both origins. The encircled region indicates the reporter allele in orientation 1 showing dATP incorporation opposite 8-oxo-G during lagging-strand replication. (c) As in panel (b), but showing the ratio of HAP-induced reversion frequencies. The encircled region depicts dHAPTP incorporation opposite template C during leading-strand replication. Figure reproduced, with permission, from Ref. [11].