Chromatin Controls DNA Replication Origin Selection, Lagging-Strand Synthesis, and Replication Fork Rates

Kurat, Christoph F.; Yeeles, Joseph T.P.; Patel, Harshil; Early, Anne; Diffley, John F.X.

doi:10.1016/j.molcel.2016.11.016

Cited by 224 publications

(286 citation statements)

References 57 publications

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“…Even at the highest concentration of Pol α tested, lagging-strand products were longer than the ∼165 nucleotides that have been measured in vivo (Smith and Whitehouse, 2012). These experiments were conducted on naked DNA templates, and in the accompanying manuscript, we show that chromatin profoundly affects lagging-strand product sizes (Kurat et al., 2016). …”

Section: Discussionmentioning

confidence: 78%

How the Eukaryotic Replisome Achieves Rapid and Efficient DNA Replication

et al. 2017

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SummaryThe eukaryotic replisome is a molecular machine that coordinates the Cdc45-MCM-GINS (CMG) replicative DNA helicase with DNA polymerases α, δ, and ε and other proteins to copy the leading- and lagging-strand templates at rates between 1 and 2 kb min−1. We have now reconstituted this sophisticated machine with purified proteins, beginning with regulated CMG assembly and activation. We show that replisome-associated factors Mrc1 and Csm3/Tof1 are crucial for in vivo rates of replisome progression. Additionally, maximal rates only occur when DNA polymerase ε catalyzes leading-strand synthesis together with its processivity factor PCNA. DNA polymerase δ can support leading-strand synthesis, but at slower rates. DNA polymerase δ is required for lagging-strand synthesis, but surprisingly also plays a role in establishing leading-strand synthesis, before DNA polymerase ε engagement. We propose that switching between these DNA polymerases also contributes to leading-strand synthesis under conditions of replicative stress.

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Section: Discussionmentioning

confidence: 78%

How the Eukaryotic Replisome Achieves Rapid and Efficient DNA Replication

et al. 2017

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“…S1B), yielding a diploid strain that produces yeast CMG. A similar method was recently used to produce the 15-member Ino80 complex (40). Purification strategies involved FLAG affinity [to capture FLAG-Mcm3/Cdc45 internal FLAG , strain yJCZ2 (Fig.…”

Section: Resultsmentioning

confidence: 99%

CMG–Pol epsilon dynamics suggests a mechanism for the establishment of leading-strand synthesis in the eukaryotic replisome

Zhou

Janska

Goswami

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

109

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The replisome unwinds and synthesizes DNA for genome duplication. In eukaryotes, the Cdc45-MCM-GINS (CMG) helicase and the leading-strand polymerase, Pol epsilon, form a stable assembly. The mechanism for coupling DNA unwinding with synthesis is starting to be elucidated, however the architecture and dynamics of the replication fork remain only partially understood, preventing a molecular understanding of chromosome replication. To address this issue, we conducted a systematic single-particle EM study on multiple permutations of the reconstituted CMG-Pol epsilon assembly. Pol epsilon contains two flexibly tethered lobes. The noncatalytic lobe is anchored to the motor of the helicase, whereas the polymerization domain extends toward the side of the helicase. We observe two alternate configurations of the DNA synthesis domain in the CMG-bound Pol epsilon. We propose that this conformational switch might control DNA template engagement and release, modulating replisome progression.DNA replication | CMG helicase | DNA polymerase | single-particle electron microscopy D NA replication is catalyzed by the replisome, a molecular machine that coordinates DNA unwinding and synthesis (1). These two functions must be tightly coordinated to prevent the rise of genome instability, which is a major cause of cancer. DNA unwinding by a replicative helicase involves single-strand translocation of a hexameric motor, whereas DNA synthesis requires template priming by a primase and extension by dedicated replicative DNA polymerases (2). In eukaryotes, the helicase function is performed by the Cdc45-MCM-GINS (CMG) complex (3, 4) and the primase function is played by Pol alpha (5), whereas DNA synthesis is catalyzed by two specialized DNA polymerases, Pol epsilon and delta. According to the consensus view, Pol epsilon synthesizes the leading and Pol delta the lagging strand (6-11). However, recent studies indicate that the division of labor between replicative polymerases might be more promiscuous than originally thought (12, 13). In in vitroreconstituted DNA replication reactions, Pol delta can support leading-strand duplication (11, 14), but switching from Pol delta to epsilon is necessary for efficient establishment of leadingstrand synthesis (14). The mechanism of substrate handoff between the two polymerases is currently unknown.Recent breakthroughs in structural biology begin to provide an architectural framework to understand the interaction between helicase and polymerases at the replication fork. For example, studies on the CMG helicase and its subcomplexes have established that the MCM is a six-member ring with an N-terminal domain that serves as a processivity collar (15) and a C-terminal ATPase motor domain that provides the DNA unwinding function (16-21). High-resolution cryo-EM analysis has shown that the ATPase motor translocates on the leading-strand template (22), in agreement with work on Xenopus embryo extracts (23). The GINS and Cdc45 components of the CMG bind to the side and stabilize the N-terminal domain of the...

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“…Compared to the S-phase-extract-based assay, ISW1a templates showed reduced replication using the fully-reconstituted assay (Figure 4—figure supplement 1A), most likely due to a lack of CREs and histone chaperones present in the S-phase-extract-based assay (Devbhandari et al, 2017; Kurat et al, 2017). Nevertheless, replication of the SWI/SNF and RSC templates was similarly reduced relative to their ISW1a-remodeled counterpart using the reconstituted assay (Figure 4B, Figure 4—figure supplement 1B and Figure 4—figure supplement 1—source data 1).…”

Section: Resultsmentioning

confidence: 99%

Nucleosomes influence multiple steps during replication initiation

Azmi

Watanabe

Maloney

et al. 2017

eLife

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Eukaryotic replication origin licensing, activation and timing are influenced by chromatin but a mechanistic understanding is lacking. Using reconstituted nucleosomal DNA replication assays, we assessed the impact of nucleosomes on replication initiation. To generate distinct nucleosomal landscapes, different chromatin-remodeling enzymes (CREs) were used to remodel nucleosomes on origin-DNA templates. Nucleosomal organization influenced two steps of replication initiation: origin licensing and helicase activation. Origin licensing assays showed that local nucleosome positioning enhanced origin specificity and modulated helicase loading by influencing ORC DNA binding. Interestingly, SWI/SNF- and RSC-remodeled nucleosomes were permissive for origin licensing but showed reduced helicase activation. Specific CREs rescued replication of these templates if added prior to helicase activation, indicating a permissive chromatin state must be established during origin licensing to allow efficient origin activation. Our studies show nucleosomes directly modulate origin licensing and activation through distinct mechanisms and provide insights into the regulation of replication initiation by chromatin.DOI: http://dx.doi.org/10.7554/eLife.22512.001

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Chromatin Controls DNA Replication Origin Selection, Lagging-Strand Synthesis, and Replication Fork Rates

Cited by 224 publications

References 57 publications

How the Eukaryotic Replisome Achieves Rapid and Efficient DNA Replication

How the Eukaryotic Replisome Achieves Rapid and Efficient DNA Replication

CMG–Pol epsilon dynamics suggests a mechanism for the establishment of leading-strand synthesis in the eukaryotic replisome

Nucleosomes influence multiple steps during replication initiation

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