Prim-pol is a recently identified DNA primase-polymerase belonging to the archaeao-eukaryotic primase (AEP) superfamily. Here, we characterize a previously unrecognized prim-pol in human cells, which we designate hPrimpol1 (human primasepolymerase 1). hPrimpol1 possesses primase and DNA polymerase activities in vitro, interacts directly with RPA1 and is recruited to sites of DNA damage and stalled replication forks in an RPA1-dependent manner. Cells depleted of hPrimpol1 display increased spontaneous DNA damage and defects in the restart of stalled replication forks. Both RPA1 binding and the primase activity of hPrimpol1 are required for its cellular function during DNA replication. Our results indicate that hPrimpol1 is a novel factor involved in the response to DNA replication stress.

The genetic cause for approximately 80% of familial breast cancer patients is unknown. Here, by sequencing the entire exomes of nine early-onset familial breast cancer patients without BRCA1/2 mutations (diagnosed with breast cancer at or before the age of 35) we found that two index cases carried a potentially deleterious mutation in the RECQL gene (RecQ helicase-like; chr12p12). Recent studies suggested that RECQL is involved in DNA double-strand break repair and it plays an important role in the maintenance of genomic stability. Therefore, we further screened the RECQL gene in an additional 439 unrelated familial breast cancer patients. In total, we found three nonsense mutations leading to a truncated protein of RECQL (p.L128X, p.W172X, and p.Q266X), one mutation affecting mRNA splicing (c.395-2A>G), and five missense mutations disrupting the helicase activity of RECQL (p.A195S, p.R215Q, p.R455C, p.M458K, and p.T562I), as evaluated through an in vitro helicase assay. Taken together, 9 out of 448 BRCA-negative familial breast cancer patients carried a pathogenic mutation of the RECQL gene compared with one of the 1,588 controls (P = 9.14×10-6). Our findings suggest that RECQL is a potential breast cancer susceptibility gene and that mutations in this gene contribute to familial breast cancer development.

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 to 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.

Background:The origin of eukaryotic histone modification enzymes still remains obscure. Results: Prototypic KMT4/Dot1 from Archaea targets chromatin proteins (Sul7d and Cren7) and shows increased activity on Sul7d, but not Cren7, in the presence of DNA. Conclusion: Promiscuous aKMT4 could be regulated by chromatin environment. Significance: This study supports the prokaryotic origin model of eukaryotic histone methyltransferases and sheds light on chromatin dynamics in Archaea.

The eukaryotic replisome is rapidly disassembled during DNA replication termination. In metazoa, the cullin‐RING ubiquitin ligase CUL‐2LRR‐1 drives ubiquitylation of the CMG helicase, leading to replisome disassembly by the p97/CDC‐48 “unfoldase”. Here, we combine in vitro reconstitution with in vivo studies in Caenorhabditis elegans embryos, to show that the replisome‐associated TIMELESS‐TIPIN complex is required for CUL‐2LRR‐1 recruitment and efficient CMG helicase ubiquitylation. Aided by TIMELESS‐TIPIN, CUL‐2LRR‐1 directs a suite of ubiquitylation enzymes to ubiquitylate the MCM‐7 subunit of CMG. Subsequently, the UBXN‐3 adaptor protein directly stimulates the disassembly of ubiquitylated CMG by CDC‐48_UFD‐1_NPL‐4. We show that UBXN‐3 is important in vivo for replisome disassembly in the absence of TIMELESS‐TIPIN. Correspondingly, co‐depletion of UBXN‐3 and TIMELESS causes profound synthetic lethality. Since the human orthologue of UBXN‐3, FAF1, is a candidate tumour suppressor, these findings suggest that manipulation of CMG disassembly might be applicable to future strategies for treating human cancer.