Chromatin Constrains the Initiation and Elongation of DNA Replication

Abstract

SUMMARY Eukaryotic chromosomal DNA is faithfully replicated in a complex series of cell-cycle-regulated events that are incompletely understood. Here we report the reconstitution of DNA replication free in solution with purified proteins from the budding yeast Saccharomyces cerevisiae. The system recapitulates regulated bidirectional origin activation; synthesis of leading and lagging strands by the three replicative DNA polymerases Pol α, Pol δ, and Pol ε; and canonical maturation of Okazaki fragments into continuous daughter strands. We uncover a dual regulatory role for chromatin during DNA replication: promoting origin dependence and determining Okazaki fragment length by restricting Pol δ progression. This system thus provides a functional platform for the detailed mechanistic analysis of eukaryotic chromosome replication.

Checkpoint Kinase Rad53 Couples Leading- and Lagging-Strand DNA Synthesis under Replication Stress

“…Therefore, we analyzed the DNA synthesis activity of Pol ε and Pol δ at different dNTP concentrations using a recently reconstituted origin-dependent budding yeast in vitro DNA replication system (Devbhandari et al, 2017). In this system, primer extension by Pol α is restricted by DNA polymerase clamp loader and clamp (RFC/PCNA), while both Pol ε and Pol δ can contribute to the synthesis of leading and lagging strands.…”

“…Reconstituted DNA replication assays were performed on chromatinized plasmid pARS1 essentially as described (Devbhandari et al, 2017) in the absence of Fen1 and Cdc9. In brief, chromatin assembly was carried out for 1 hour at 30°C in a 10μl reaction volume including 3.1 μM Nap1, 350 nM histone octamer, 50 nM Isw1a, 20 nM pARS1 (4.8 kb), and 100 nM ORC; to induce Mcm2-7 loading the chromatin assembly mix was subsequently supplemented with 50 nM Cdc6 and 100 nM Cdt1•Mcm2-7, followed by further incubation for 30 minutes at 30°C.…”

From structure to mechanism—understanding initiation of DNA replication

“…Initial priming of DNA synthesis is carried out by DNA polymerase α, while leading and lagging strand DNA synthesis occurs mainly via polymerase ε (Pursell et al 2007;Burgers et al 2016) and polymerase δ (Nick McElhinny et al 2008), respectively. However, some plasticity exists, with polymerase δ also playing a role during leading strand DNA synthesis, particularly during initiation of DNA replication and under conditions of replicative stress in budding yeast (Pavlov et al 2001;Devbhandari et al 2017;Yeeles et al 2017) and after homologous recombination-dependent fork restart in Schizosaccharomyces pombe (Miyabe et al 2015). Mcm10 acts also during elongation, as it travels with the replisome (Ricke and Bielinsky 2004;Gambus et al 2006;Pacek et al 2006), and as a specific mutation in its C terminus results in shorter replication products without affecting origin firing (Looke et al 2017).…”

“…Sequence-specific recognition of DNA replication origins by the S. cerevisiae ORC formed the biochemical basis for the discovery of this important DNA replication factor in 1992 (Bell and Stillman 1992), which started a 25-year journey toward the full reconstitution of budding yeast DNA replication (Yeeles et al 2015(Yeeles et al , 2017Devbhandari et al 2017). The six-subunit Orc1-6 complex is well conserved from yeast to humans, and this homology extends even to archaea, where Orc monomers or dimers function in origin recognition .…”

“…In vivo analysis has identified most key players and addressed many regulatory principles, but many crucial mechanisms remain unknown. The recent reconstitution of budding yeast DNA replication using purified proteins now offers the chance for a detailed mechanistic and structural analysis of DNA replication initiation and DNA synthesis, which will help to advance the entire field (Yeeles et al 2015(Yeeles et al , 2017Devbhandari et al 2017). …”