Excess MCM proteins protect human cells from replicative stress by licensing backup origins of replication

Abstract

The six main minichromosome maintenance proteins (Mcm2-7), which presumably constitute the core of the replicative DNA helicase, are present in chromatin in large excess relative to the number of active replication forks. To evaluate the relevance of this apparent surplus of Mcm2-7 complexes in human cells, their levels were down-regulated by using RNA interference. Interestingly, cells continued to proliferate for several days after the acute (>90%) reduction of Mcm2-7 concentration. However, they became hypersensitive to DNA replication stress, accumulated DNA lesions, and eventually activated a checkpoint response that prevented mitotic division. When this checkpoint was abrogated by the addition of caffeine, cells quickly lost viability, and their karyotypes revealed striking chromosomal aberrations. Singlemolecule analyses revealed that cells with a reduced concentration of Mcm2-7 complexes display normal fork progression but have lost the potential to activate ''dormant'' origins that serve a backup function during DNA replication. Our data show that the chromatin-bound ''excess'' Mcm2-7 complexes play an important role in maintaining genomic integrity under conditions of replicative stress.DNA combing ͉ DNA replication ͉ origin licensing R apidly proliferating cells start to prepare for DNA replication several hours before the actual S-phase, with the assembly of prereplication complexes (pre-RCs) at origins in telophase and early G 1 . Pre-RC assembly, also referred to as ''origin licensing,'' consists in the recruitment of Mcm2-7 protein complexes by initiator proteins ORC, CDC6, and CDT1. ORC and CDC6 likely constitute a structural module with ATPase activity that opens and closes the ring-shaped MCM hexamer, facilitating its topological engagement with the DNA, whereas CDT1 cooperates in the loading reaction as a molecular chaperone (reviewed in refs. 1 and 2). Different lines of evidence indicate that Mcm2-7 constitute the core of the replicative DNA helicase in eukaryotic cells in association with CDC45 and the GINS complex (3, 4).The maximum number of origins available in the subsequent S-phase is predetermined at the licensing stage, because additional pre-RCs cannot be assembled later in the cell cycle because of the inhibitory activity of the S, G 2 and M-phase cyclin-dependent kinases. This regulation establishes a temporal alternation of origin licensing and firing that is important to prevent DNA overreplication. In yeast, blending the licensing and firing periods by deletion of the CDK inhibitor Sic1 or by overexpression of the G 1 cyclin Cln2, greatly increased genomic instability (5, 6). In human cells, premature expression of Cyclin E during mitosis and G 1 interfered with the association of MCM proteins with chromatin and at the same time promoted the firing of the limited number of licensed origins, effectively accelerating the G 1 -S transition (7). Nevertheless, cells continued to proliferate under these challenging conditions and accumulated karyotypic defects (8). These results are ...

Defining replication origin efficiency using DNA fiber assays

“…These include overexpression of translesion DNA polymerases (Pillaire et al 2007), the depletion of CHK1 (Maya-Mendoza et al 2007), BLM (Rao et al 2007), HR proteins (Daboussi et al 2008), and topoisomerase I (Tuduri et al 2009) and exposure to HU (Ge et al 2007;Ibarra et al 2008). Importantly, the latter studies also revealed that dormant origins are dispensable for normal growth but are essential for viability under replication stress (Ge et al 2007;Ibarra et al 2008).…”

“…Replication origins are also present in large excess in higher eukaryotes. Most of these so-called dormant origins are not used in normal growth conditions but fire when DNA replication is challenged with genotoxic drugs (Ge et al 2007;Ibarra et al 2008). Origin redundancy appears therefore an important mechanism preventing genomic instability during S phase.…”

Chk1–cyclin A/Cdk1 axis regulates origin firing programs in mammals

“…Instead, origin firing is likely dependent on stochastic initiation at dormant origins defined as such be their chromatin status (Gilbert 2004), despite the presence of some origins that are sequence specific (Huberman 1998). Stochastic initiation is organized by feedback regulation from active replicons, which is mediated by the sensing of ongoing replication (Ge et al 2007;Ibarra et al 2008). In the Xenopus laevis egg system, these ongoing replication events appear to be sensed by the DNA-damage checkpoint kinases ataxia telangiectasia mutated (ATM) and/or AT and Rad3 related (ATR) because inhibition of ATM and ATR with caffeine or specific neutralizing antibodies promoted rapid and synchronous origin firing (Shechter et al 2004).…”

Too much to handle — how gaining chromosomes destabilizes the genome

“…Interestingly, MCM2-7 helicase is loaded in excess on chromatin; unused, dormant origins can become activated to finish replication when the active replication forks get stalled. [26][27][28] As a consequence, partial depletion of MCM2-7 under normal conditions is not detrimental for cells as they do not need to activate dormant origins, while it becomes a problem when DNA replication is challenged, as there are no additional origins to be fired and rescue the stalled DNA replication. 27,28 By ensuring timely DNA replication under conditions of replication stress, the excess MCM2-7 in cells is crucial for maintenance of genome stability.…”