Mitotic CDK promotes replisome disassembly, fork breakage, and complex DNA rearrangements

Abstract

22DNA replication errors generate complex chromosomal rearrangements and thereby 23 contribute to tumorigenesis and other human diseases. Although the events that trigger 24 these errors are not well understood, one candidate is mitotic entry before the 25 completion of DNA replication. To address the impact of mitosis on DNA replication, we 26 employed Xenopus egg extracts. When mitotic CDK (Cyclin B1-CDK1) is used to drive 27 these extracts into mitosis, the E3 ubiquitin ligase TRAIP promotes ubiquitylation of the 28 replicative CMG (CDC45/MCM2-7/GINS) helicase at stalled forks and at forks that have 29 completed DNA synthesis. In both cases, ubiquitylation is followed by CMG extraction 30 from chromatin by the CDC48/p97 ATPase. At stalled forks, CMG removal results in 31 fork breakage and complex end joining events involving deletions and template-32 switching. Our results identify TRAIP-dependent replisome disassembly as a novel 33 trigger of replication fork collapse and propose it underlies complex DNA 34 rearrangements in mitosis. 35 3 HIGHLIGHTS 36 1. TRAIP-dependent MCM7 ubiquitylation removes all CMGs from chromatin in 37 mitosis 38 2. CMG unloading from stalled forks causes replication fork breakage 39 3. Replication fork breakage in mitosis causes complex rearrangements 40 4. New model of replication fork collapse 41 42 43Genome evolution occurs through the gradual accrual of genetic changes or in a 44 saltatory manner, with bursts of chromosomal alterations originating from single 45 catastrophic events (Holland and Cleveland, 2012; Leibowitz et al., 2015; Liu et al., 46 2011; Stephens et al., 2011). Many chromosomal alterations can be traced to DNA 47 breaks that arise during DNA replication (Hills and Diffley, 2014; Mankouri et al., 2013; 48 Techer et al., 2017). However, there is an ongoing debate about when and how 49 replication fork breakage is triggered (Toledo et al., 2017). 50In normal cells, multiple cell cycle regulatory controls and error correction 51 mechanisms prevent DNA replication errors (Hills and Diffley, 2014). Cells prepare for 52 DNA replication in the G1 phase of the cell cycle, when pairs of MCM2-7 ATPases are 53 recruited to each origin ("licensing"). In S phase, cyclin-dependent kinase (CDK) 54 promotes the association of CDC45 and GINS with MCM2-7, leading to formation of the 55 replicative CMG helicase complex (CDC45-MCM2-7-GINS) ("initiation"). CMG 56 unwinding of the origin nucleates the assembly of two DNA replication forks that travel 57 away from the origin, copying DNA as they go ("elongation"). When converging forks 58 from adjacent origins meet, the replisome is disassembled ("termination"). Replisome 59 disassembly in S phase requires the E3 ubiquitin ligase CRL2 Lrr1 , which ubiquitylates 60 the MCM7 subunit of CMG, leading to CMG's extraction from chromatin by the p97 61 ATPase (Dewar et al., 2017; Sonneville et al., 2017). In the absence of CRL2 Lrr1 , CMGs 62 persist on chromatin until mitosis, but are then removed by a secondary, p97-dependent ...

CUL2LRR1, TRAIP and p97 control CMG helicase disassembly in the mammalian cell cycle

“…Importantly, the TRUL‐1 / TRAIP pathway of mitotic CMG disassembly in C. elegans and X. laevis is activated by mitosis but does not require DNA replication termination. Thus, CMG disassembly still occurs if C. elegans cells enter mitosis before the completion of DNA replication (Sonneville et al , 2019), and the same is true if Xenopus egg extracts are induced to enter mitosis with incompletely replicated DNA (Deng et al , 2019; Priego Moreno et al , 2019). Loss of TRUL‐1 in C. elegans causes reduced viability in combination with a mutation impairing DNA replication (Sonneville et al , 2019), suggesting that the mitotic CMG disassembly pathway evolved to process any remaining sites of incomplete DNA replication before anaphase, in order to facilitate the preservation of genome integrity in dividing cells.…”

Small but strong: Mutational and functional landscapes of micronuclei in cancer genomes

“…Cells with chromosomal loci that are hard to replication (eg, common fragile sites) often fail to complete replication during interphase and instead enter mitosis with unreplicated regions that will be further replicated via a process known as mitotic DNA repair synthesis 84,85 . During this process, mitotic CDK activates the E3 ubiquitin ligase TRAIP, which drives replisome disassembly and thereby provides access to replication forks for other factors to finish replication 86,87 . Similarly, the incompletely replicated regions of micronucleus also undergo mitotic replication when the main nucleus enters mitosis 32 .…”

“…Similarly, the incompletely replicated regions of micronucleus also undergo mitotic replication when the main nucleus enters mitosis 32 . Since micronucleus typically possesses multiple unreplicated loci, massive DNA fragmentation would be expected in principle 32,86 . Indeed, enforcing micronucleus into mitosis by mitotic main nucleus produces ssDNA and DSBs 32 .…”

Preparation for DNA replication: the key to a successful S phase

“…MCM complexes in active CMG complexes at replication forks are removed during replisome disassembly. Two proposed mechanisms have been reported: (a) association with an alternate Mcm2 subunit, MCM‐BP, has been suggested to unload MCM2‐7 from DNA during S phase (though MCM‐BP has many partners and proposed roles), and (b) polyubiquitylation of the Mcm7 subunit stimulates Mcm7 removal from the complex by a nondegradative mechanism promoting complete replisome disassembly and reviewed in . However, many questions remain about the mechanisms and order of events of MCM unloading at the end of S phase.…”