Evidence obtained from studies with yeast and Xenopus indicate that the initiation of DNA replication is a multistep process. The origin recognition complex (ORC), Cdc6p, and minichromosome maintenance (MCM) proteins are required for establishing prereplication complexes, upon which initiation is triggered by the activation of cyclin-dependent kinases and the Dbf4p-dependent kinase Cdc7p. The identification of human homologues of these replication proteins allows investigation of S-phase regulation in mammalian cells. Using centrifugal elutriation of several human cell lines, we demonstrate that whereas human Orc2 (hOrc2p) and hMcm proteins are present throughout the cell cycle, hCdc6p levels vary, being very low in early G 1 and accumulating until cells enter mitosis. hCdc6p can be polyubiquitinated in vivo, and it is stabilized by proteasome inhibitors. Similar to the case for hOrc2p, a significant fraction of hCdc6p is present on chromatin throughout the cell cycle, whereas hMcm proteins alternate between soluble and chromatin-bound forms. Loading of hMcm proteins onto chromatin occurs in late mitosis concomitant with the destruction of cyclin B, indicating that the mitotic kinase activity inhibits prereplication complex formation in human cells.The molecular mechanism that restricts firing of origins of replication to once per cell cycle invokes the ordered binding to and/or release of different replication proteins from specific DNA sequences (replicators) located in the vicinity of the actual origins of DNA replication. Following separation of sister chromatids at mitosis and during the subsequent G 1 phase, prereplication complexes (pre-RCs) are formed at origins of DNA replication. Initiation of DNA replication is triggered by the action of at least two sets of protein kinase activities, cyclin-dependent kinases (CDKs) and Dbf4p-Cdc7p. After initiation, the protein complex at each origin changes to a postreplication state (post-RC), thereby preventing further initiation events for the rest of the cell cycle (reviewed in references 15 and 57).The origin recognition complex (ORC), a six-subunit initiator protein (2), is present in both pre-and post-RCs (10), and one of its functions is to mark the position of replication origins in the genome. The pre-RC is established by the regulated binding of additional factors, which include Cdc6p and the minichromosome maintenance (MCM) proteins. Yeast CDC6 displays a genetic interaction with the ORC and is a critical factor for establishing the competence of replication origins once per cell cycle (12,33,47,48). Besides its function in DNA replication, it may also be involved in a mitotic checkpoint control, because Cdc6-deprived yeast cells that do not replicate DNA still undergo a reductional mitosis (4,47,63

SummaryWe describe a second primase in human cells, PrimPol, which has the ability to start DNA chains with deoxynucleotides unlike regular primases, which use exclusively ribonucleotides. Moreover, PrimPol is also a DNA polymerase tailored to bypass the most common oxidative lesions in DNA, such as abasic sites and 8-oxoguanine. Subcellular fractionation and immunodetection studies indicated that PrimPol is present in both nuclear and mitochondrial DNA compartments. PrimPol activity is detectable in mitochondrial lysates from human and mouse cells but is absent from mitochondria derived from PRIMPOL knockout mice. PRIMPOL gene silencing or ablation in human and mouse cells impaired mitochondrial DNA replication. On the basis of the synergy observed with replicative DNA polymerases Polγ and Polε, PrimPol is proposed to facilitate replication fork progression by acting as a translesion DNA polymerase or as a specific DNA primase reinitiating downstream of lesions that block synthesis during both mitochondrial and nuclear DNA replication.

Haematopoietic stem cells (HSCs) self-renew for life, thereby making them one of the few blood cells that truly age1,2. Paradoxically, although HSCs numerically expand with age, their functional activity declines over time, resulting in degraded blood production and impaired engraftment following transplantation2. While many drivers of HSC ageing have been proposed2–5, the reason why HSC function degrades with age remains unknown. Here we show that cycling old HSCs in mice have heightened levels of replication stress associated with cell cycle defects and chromosome gaps or breaks, which are due to decreased expression of mini-chromosome maintenance (MCM) helicase components and altered dynamics of DNA replication forks. Nonetheless, old HSCs survive replication unless confronted with a strong replication challenge, such as transplantation. Moreover, once old HSCs re-establish quiescence, residual replication stress on ribosomal DNA (rDNA) genes leads to the formation of nucleolar-associated γH2AX signals, which persist owing to ineffective H2AX dephosphorylation by mislocalized PP4c phosphatase rather than ongoing DNA damage. Persistent nucleolar γH2AX also acts as a histone modification marking the transcriptional silencing of rDNA genes and decreased ribosome biogenesis in quiescent old HSCs. Our results identify replication stress as a potent driver of functional decline in old HSCs, and highlight the MCM DNA helicase as a potential molecular target for rejuvenation therapies.

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 ...