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DNA replication is a prerequisite for cell proliferation, yet it can be increasingly challenging for a eukaryotic cell to faithfully duplicate its genome as its size and complexity expands. Dormant origins now emerge as a key component for cells to successfully accomplish such a demanding but essential task. In this perspective, we will first provide an overview of the fundamental processes eukaryotic cells have developed to regulate origin licensing and firing. With a special focus on mammalian systems, we will then highlight the role of dormant origins in preventing replication-associated genome instability and their functional interplay with proteins involved in the DNA damage repair response for tumor suppression. Lastly, deficiencies in the origin licensing machinery will be discussed in relation to their influence on stem cell maintenance and human diseases. [ABSTRACT FROM AUTHOR]

Summary Mcm2-7 helicase is loaded onto double-stranded origin DNA as an inactive double hexamer (DH) in G1 phase. The mechanisms of Mcm2-7 remodeling that trigger helicase activation in S phase remain unknown. Here, we develop an approach to detect and purify the endogenous DHs directly. Through cellular fractionation, we provide in vivo evidence that DHs are assembled on chromatin in G1 phase and separated during S phase. Interestingly, Mcm10, a robust MCM interactor, co-purifies exclusively with the DHs in the context of chromatin. Deletion of the main interaction domain, Mcm10 C terminus, causes growth and S phase defects, which can be suppressed through Mcm10-MCM fusions. By monitoring the dynamics of MCM DHs, we show a significant delay in DH dissolution during S phase in the Mcm10-MCM interaction-deficient mutants. Therefore, we propose an essential role for Mcm10 in Mcm2-7 remodeling through formation of a cell-cycle-regulated supercomplex with DHs. [ABSTRACT FROM AUTHOR]

Abstract: The ability of a eukaryotic cell to precisely and accurately replicate its DNA is crucial to maintain genome stability. Here we describe our current understanding of the process by which origins are licensed for DNA replication and review recent work suggesting that fork stalling has exerted a strong selective pressure on the positioning of licensed origins. In light of this, we discuss the complex and disparate phenotypes observed in mouse models and humans patients that arise due to defects in replication licensing proteins. [Copyright &y& Elsevier]

Fundamental to our understanding of chromosome duplication is the idea that replication origins function both as sites where MCM helicases are loaded during the G1 phase and where synthesis begins in S phase. However, the temporal delay between phases exposes the replisome assembly pathway to potential disruption prior to replication. Using multicolor, single-molecule imaging, we systematically study the consequences of encounters between actively transcribing RNA polymerases (RNAPs) and replication initiation intermediates in the context of chromatin. We demonstrate that RNAP can push multiple licensed MCM helicases over long distances with nucleosomes ejected or displaced. Unexpectedly, we observe that MCM helicase loading intermediates also can be repositioned by RNAP and continue origin licensing after encounters with RNAP, providing a web of alternative origin specification pathways. Taken together, our observations reveal a surprising mobility in origin-licensing factors that confers resistance to the complex challenges posed by diverse obstacles encountered on chromosomes. [Display omitted] • RNA polymerase robustly pushes MCMs without slowing down • Nucleosomes are ejected or pushed by MCMs during repositioning • RNA polymerase can bypass ORC or activate ORC sliding and OCCM sliding • Origin licensing continues after RNA polymerase encounters and relocalization Scherr et al. report a single-molecule imaging approach that provides a real-time view of encounters between RNA polymerase and replication origin-licensing factors. They show that origin-licensing factors can be pushed to new locations by RNA polymerase to support alternative origin specification pathways. [ABSTRACT FROM AUTHOR]

Abstract: MCM2–7 proteins are conserved replication factors functioning as DNA helicases during DNA synthesis. MCM8 is another member of this family, which appears to be specific for higher eukaryotes, as it is absent in worms and yeast. Here we report the complete identification of a novel member of this family, the MCM9 protein. Like MCM8, MCM9 is only present in the genome of higher eukaryotes. This protein contains an MCM8-like ATP binding and hydrolysis motif implicated in helicase activity. Strikingly, MCM9 also contains a unique carboxy-terminal domain which has only weak homology to MCM2–7 and MCM8 but is conserved within MCM9 homologs. We also show that the very recently reported human MCM9 protein (HsMCM9), which resembles a truncated MCM-like protein missing a part of the MCM2–7 signature domain, is an incomplete form of the full length HsMCM9 described here. Searching the human genome with either the newly identified human MCM9 or other MCM protein sequences, we did not detect further additional members of this DNA helicase family and suggest that it is constituted of eight members, falling into two different groups, one constituted by the MCM2–7 complex and the other by MCM8 and MCM9, which are present only in higher eukaryotes. [Copyright &y& Elsevier]

The proliferation of eukaryotic cells is a highly regulated process that depends on the precise duplication of chromosomal DNA in each cell cycle. Regulation of the replication licensing system, which promotes the assembly of complexes of proteins termed Mcm2–7 onto replication origins, is responsible for preventing re-replication of DNA in a single cell cycle. Recent work has shown how the licensing system is directly controlled by cyclin-dependent kinases (CDKs). Repression of origin licensing is emerging as a ubiquitous route by which the proliferative capacity of cells is lowered, and Mcm2–Mcm7 proteins show promise as diagnostic markers of early cancer stages. These results have prompted us to propose a functional distinction between the proliferative state and the non-proliferative state (including G0) depending on whether origins are licensed. [ABSTRACT FROM AUTHOR]