Moreno SP and Gambus A (2020), Mechanisms of eukaryotic replisome disassembly.

XB-ART-57838

Biochem Soc Trans 2020 Jun 30;483:823-836. doi: 10.1042/BST20190363.

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Mechanisms of eukaryotic replisome disassembly.

Moreno SP , Gambus A .

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DNA replication is a complex process that needs to be executed accurately before cell division in order to maintain genome integrity. DNA replication is divided into three main stages: initiation, elongation and termination. One of the key events during initiation is the assembly of the replicative helicase at origins of replication, and this mechanism has been very well described over the last decades. In the last six years however, researchers have also focused on deciphering the molecular mechanisms underlying the disassembly of the replicative helicase during termination. Similar to replisome assembly, the mechanism of replisome disassembly is strictly regulated and well conserved throughout evolution, although its complexity increases in higher eukaryotes. While budding yeast rely on just one pathway for replisome disassembly in S phase, higher eukaryotes evolved an additional mitotic pathway over and above the default S phase specific pathway. Moreover, replisome disassembly has been recently found to be a key event prior to the repair of certain DNA lesions, such as under-replicated DNA in mitosis and inter-strand cross-links (ICLs) in S phase. Although replisome disassembly in human cells has not been characterised yet, they possess all of the factors involved in these pathways in model organisms, and de-regulation of many of them are known to contribute to tumorigenesis and other pathological conditions.

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	Figure 1. Simplified model of eukaryotic DNA replication.Inactive double hexamers of the replicative helicase core Mcm2–7 are loaded onto origins at the end of mitosis/G1 phase (origin licensing). Origins are activated during S phase with the assembly of the active replicative helicase, also known as CMG (Cdc45-Mcm2–7-GINS), which involves the recruitment of GINS and Cdc45 to the Mcm2–7 complex. While double hexamers of Mcm2–7 encircle dsDNA, CMGs encircle ssDNA and translocate in an ‘N-terminus first' direction thus passing each other within the origin (origin firing). Replisomes are built around CMGs and they progress through the chromatin unwinding DNA and promoting DNA synthesis in a semi-discontinuous way (elongation). Replication forks from neighbouring origins finally converge, leading to complete replication of sister chromatids (termination).
	Figure 2. Mechanism of eukaryotic DNA replication termination.Convergence of replication forks during termination is facilitated by Pif1 family helicases, likely due to their ability to unwind DNA in the opposite direction of CMG movement. CMGs continue to translocate on the leading strand templates and pass one another while leading strands continue to be extended. CMGs encounter the last Okazaki fragments on lagging strands and switch to encircle dsDNA. The last Okazaki fragments are processed by an unknown mechanism. CMGs are disassembled only after DNA has been fully ligated.
	Figure 3. Mechanisms of eukaryotic replisome disassembly during termination.(A) Replisome disassembly during termination in Saccharomyces cerevisiae. SCFDia2 might be constitutively associated with the budding yeast replisome and promotes CMG ubiquitylation on its Mcm7 subunit during termination in the form of K48-linked ubiquitin chains. Alternatively, it is possible that SCFDia2 is recruited to the replisome only during termination, leading to Mcm7 ubiquitylation. CMG ubiquitylation triggers Cdc48-Ufd1-Npl4-dependent recruitment and replisome disassembly. (B) Replisome disassembly during termination in higher eukaryotes. DNA replication termination promotes de novo recruitment of Cullin2LRR1 to the terminated CMGs, which results in the ubiquitylation of Mcm7 with K48-linked ubiquitin chains. The ubiquitylated replisome is subsequently disassembled by p97-Ufd1-Npl4.
	Figure 4. TRAIP-dependent replisome disassembly pathways.(A) Disassembly of terminated replisomes in mitosis. When termination occurs in the absence of Cullin2LRR1 activity, the terminated replisomes remain associated with chromatin during S phase. Entry into mitosis, driven by an increase in CDK activity, promotes TRAIP-dependent ubiquitylation of Mcm7 within terminated CMGs with K6/K63-linked ubiquitin chains. The ubiquitylated terminated replisome is finally disassembled by p97-Ufd1-Npl4. (B) Disassembly of stalled replisomes in mitosis required for the processing of under-replicated DNA. When DNA replication proceeds in the presence of replication stress-inducing agents such as aphidicolin, replication forks slow down and compromise forks convergence during S phase, thus increasing the risk of entering mitosis with under-replicated DNA. When mitosis ensues in the presence of under-replicated DNA, TRAIP promotes ubiquitylation of Mcm7 within stalled CMGs with long ubiquitin chains, followed by disassembly of the stalled replisomes by p97-Ufd1-Npl4 and DNA incisions in the leading strand templates. In this scenario, one of the chromatids is faithfully repaired through gap filling (1) while repair of the other one requires the joining of the two broken ends likely through the alt-NHEJ mechanisms of SSA and MMEJ. This chromatid, therefore, receives a deletion encompassing the under-replicated DNA and exhibits sister chromatid exchange (SCE) and potential templated insertions (2). (C) Disassembly of replisomes prior to ICL repair. Convergence of replication forks at both sites of the ICL triggers ubiquitylation of several CMG components by TRAIP. These ubiquitin chains are long and heterotypically linked and/or branched. Ubiquitylated CMGs are disassembled by p97, followed by fork reversal, nucleolytic incisions, ICL unhooking and DNA repair. One of the chromatids is repaired through translesion synthesis (1) while the other one is repaired through homologous recombination (2).

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Alabert, Chromatin Replication and Histone Dynamics. 2017, Pubmed