Abstract
We have reconstituted a eukaryotic leading/lagging strand replisome comprising 31 distinct polypeptides. This study identifies a process unprecedented in bacterial replisomes. While bacteria and phage simply recruit polymerases to the fork, we find that suppression mechanisms are used to position the distinct eukaryotic polymerases on their respective strands. Hence, Pol ε is active with CMG on the leading strand, but it is unable to function on the lagging strand, even when Pol δ is not present. Conversely, Pol δ-PCNA is the only enzyme capable of extending Okazaki fragments in the presence of Pols ε and α. We have shown earlier that Pol δ-PCNA is suppressed on the leading strand with CMG (Georgescu et al., 2014). We propose that CMG, the 11-subunit helicase, is responsible for one or both of these suppression mechanisms that spatially control polymerase occupancy at the fork.DOI: http://dx.doi.org/10.7554/eLife.04988.001
Highlights
Composition of the eukaryotic replisome and the function of its various proteins is an area of active investigation
We demonstrate here that Pol δ is active on the lagging strand in the presence of Pol ε and Pol α
The discontinuous lagging strand is a more difficult process than continuous leading strand synthesis, and the current study aims to identify how polymerases are coordinated during coupled leading–lagging strand synthesis
Summary
Composition of the eukaryotic replisome and the function of its various proteins is an area of active investigation. Cellular studies reveal that eukaryotes use two different DNA polymerases for the leading and lagging strands, Pols ε and δ, respectively (Lee et al, 1989; Weinberg and Kelly, 1989; Tsurimoto et al, 1990; Waga and Stillman, 1998; Benkovic et al, 2001; Pursell et al, 2007; Kunkel and Burgers, 2008; Nick McElhinny et al, 2008; Stillman, 2008). Numerous other proteins travel with eukaryotic replication forks and have no bacterial homolog or known function. Many replication fork-associated proteins undergo modifications in response to the cell cycle or DNA damage
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