The genomic architecture of the PROS1 gene underlying large tandem duplication mutation that causes thrombophilia from hereditary protein S deficiency

Hereditary protein S deficiency from a mutation in the PROS1 gene causes a genetic predisposition to develop venous thromboembolic disorders in humans. Recently, the acknowledgment of the clinical significance of large copy number mutations in protein S deficiency has increased. In this study, the authors investigated the genomic architecture of PROS1 in order to understand the microscopic sequence environment leading to large intragenic copy number mutations in the gene. The study subjects were 3 unrelated male patients with hereditary protein S deficiency from a tandem duplication mutation involving exons 5–10 of PROS1. Breakpoint analyses revealed 10-bp microhomology sequences in the intervening sequence (IVS)-4 and IVS-10 at the duplication junction without additional sequence changes, suggesting a single replication-based event as the potential molecular mechanism of rearrangement and founder effect in the mutant alleles. Further analyses on nucleotide sequences flanking the microhomology sequence revealed the presence of a repeat element (LTR-ERV1) and quadruplex-forming G-rich sequences in IVS-4. The results from genotyping multi-allelic short tandem repeats supported founder effect in the identical mutations in the 3 unrelated patients. In conclusion, we identified unique genomic architectures in the intervening sequences of PROS1 that underlie a large intragenic tandem duplication mutation leading to inherited thrombophilia.     

Tel1/ATM prevents degradation of replication forks that reverse after topoisomerase poisoning

In both yeast and mammals, the topoisomerase poison camptothecin (CPT) induces fork reversal, which has been proposed to stabilize replication forks, thus providing time for the repair of CPT‐induced lesions and supporting replication restart. We show that Tel1, the Saccharomyces cerevisiae orthologue of human ATM kinase, stabilizes CPT‐induced reversed forks by counteracting their nucleolytic degradation by the MRX complex. Tel1‐lacking cells are hypersensitive to CPT specifically and show less reversed forks in the presence of CPT. The lack of Mre11 nuclease activity restores wild‐type levels of reversed forks in CPT‐treated tel1Δ cells without affecting fork reversal in wild‐type cells. Moreover, Mrc1 inactivation prevents fork reversal in wild‐type, tel1Δ, and mre11 nuclease‐deficient cells and relieves the hypersensitivity of tel1Δ cells to CPT. Altogether, our data indicate that Tel1 counteracts Mre11 nucleolytic activity at replication forks that undergo Mrc1‐mediated reversal in the presence of CPT.     

Cycling with BRCA2 from DNA repair to mitosis

Genetic integrity in proliferating cells is guaranteed by the harmony of DNA replication, appropriate DNA repair, and segregation of the duplicated genome. Breast cancer susceptibility gene BRCA2 is a unique tumor suppressor that is involved in all three processes. Hence, it is critical in genome maintenance. The functions of BRCA2 in DNA repair and homology-directed recombination (HDR) have been reviewed numerous times. Here, I will briefly go through the functions of BRCA2 in HDR and focus on the emerging roles of BRCA2 in telomere homeostasis and mitosis, then discuss how BRCA2 exerts distinct functions in a cell-cycle specific manner in the maintenance of genomic integrity.     

CDK targeting of NBS1 promotes DNA-end resection, replication restart and homologous recombination

The conserved MRE11–RAD50–NBS1 (MRN) complex is an important sensor of DNA double-strand breaks (DSBs) and facilitates DNA repair by homologous recombination (HR) and end joining. Here, we identify NBS1 as a target of cyclin-dependent kinase (CDK) phosphorylation. We show that NBS1 serine 432 phosphorylation occurs in the S, G2 and M phases of the cell cycle and requires CDK activity. This modification stimulates MRN-dependent conversion of DSBs into structures that are substrates for repair by HR. Impairment of NBS1 phosphorylation not only negatively affects DSB repair by HR, but also prevents resumption of DNA replication after replication-fork stalling. Thus, CDK-mediated NBS1 phosphorylation defines a molecular switch that controls the choice of repair mode for DSBs.     

EXO5-DNA structure and BLM interactions direct DNA resection critical for ATR-dependent replication restart

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RNase H eliminates R‐loops that disrupt DNA replication but is nonessential for efficient DSB repair

In Saccharomyces cerevisiae, genome stability depends on RNases H1 and H2, which remove ribonucleotides from DNA and eliminate RNA–DNA hybrids (R‐loops). In Schizosaccharomyces pombe, RNase H enzymes were reported to process RNA–DNA hybrids produced at a double‐strand break (DSB) generated by I‐PpoI meganuclease. However, it is unclear if RNase H is generally required for efficient DSB repair in fission yeast, or whether it has other genome protection roles. Here, we show that S. pombe rnh1? rnh201? cells, which lack the RNase H enzymes, accumulate R‐loops and activate DNA damage checkpoints. Their viability requires critical DSB repair proteins and Mus81, which resolves DNA junctions formed during repair of broken replication forks. “Dirty” DSBs generated by ionizing radiation, as well as a “clean” DSB at a broken replication fork, are efficiently repaired in the absence of RNase H. RNA–DNA hybrids are not detected at a reparable DSB formed by fork collapse. We conclude that unprocessed R‐loops collapse replication forks in rnh1? rnh201? cells, but RNase H is not generally required for efficient DSB repair.     

DNA instability at chromosomal fragile sites in cancer

Human chromosomal fragile sites are specific genomic regions which exhibit gaps or breaks on metaphase chromosomes following conditions of partial replication stress. Fragile sites often coincide with genes that are frequently rearranged or deleted in human cancers, with over half of cancer-specific translocations containing breakpoints within fragile sites. But until recently, little direct evidence existed linking fragile site breakage to the formation of cancer-causing chromosomal aberrations. Studies have revealed that DNA breakage at fragile sites can induce formation of RET/PTC rearrangements, and deletions within the FHIT gene, resembling those observed in human tumors. These findings demonstrate the important role of fragile sites in cancer development, suggesting that a better understanding of the molecular basis of fragile site instability is crucial to insights in carcinogenesis. It is hypothesized that under conditions of replication stress, stable secondary structures form at fragile sites and stall replication fork progress, ultimately resulting in DNA breaks. A recent study examining an FRA16B fragment confirmed the formation of secondary structure and DNA polymerase stalling within this sequence in vitro, as well as reduced replication efficiency and increased instability in human cells. Polymerase stalling during synthesis of FRA16D has also been demonstrated. The ATR DNA damage checkpoint pathway plays a critical role in maintaining stability at fragile sites. Recent findings have confirmed binding of the ATR protein to three regions of FRA3B under conditions of mild replication stress. This review will discuss recent advances made in understanding the role and mechanism of fragile sites in cancer development.     

DNA Polymerases that Propagate the Eukaryotic DNA Replication Fork

Abstract

Three DNA polymerases are thought to function at the eukaryotic DNA replication fork. Currently, a coherent model has been derived for the composition and activities of the lagging strand machinery. RNA-DNA primers are initiated by DNA polymerase α -primase. Loading of the proliferating cell nuclear antigen, PCNA, dissociates DNA polymerase α and recruits DNA polymerase δ and the flap endonuclease FEN1 for elongation and in preparation for its requirement during maturation, respectively. Nick translation by the strand displacement action of DNA polymerase δ, coupled with the nuclease action of FEN1, results in processive RNA degradation until a proper DNA nick is reached for closure by DNA ligase I. In the event of excessive strand displacement synthesis, other factors, such as the Dna2 nuclease/helicase, are required to trim excess flaps. Paradoxically, the composition and activity of the much simpler leading strand machinery has not been clearly established. The burden of evidence suggests that DNA polymerase ε normally replicates this strand, but under conditions of dysfunction, DNA polymerase δ may substitute.     

Starfish: Fault-Tolerant Dynamic MPI Programs on Clusters of Workstations

This paper reports on the architecture and design of Starfish, an environment for executing dynamic (and static) MPI-2 programs on a cluster of workstations. Starfish is unique in being efficient, fault-tolerant, highly available, and dynamic as a system internally, and in supporting fault-tolerance and dynamicity for its application programs as well. Starfish achieves these goals by combining group communication technology with checkpoint/restart, and uses a novel architecture that is both flexible and portable and keeps group communication outside the critical data path, for maximum performance.     

Separation of intra-S checkpoint protein contributions to DNA replication fork protection and genomic stability in normal human fibroblasts

The ATR-dependent intra-S checkpoint protects DNA replication forks undergoing replication stress. The checkpoint is enforced by ATR-dependent phosphorylation of CHK1, which is mediated by the TIMELESS-TIPIN complex and CLASPIN. Although loss of checkpoint proteins is associated with spontaneous chromosomal instability, few studies have examined the contribution of these proteins to unchallenged DNA metabolism in human cells that have not undergone carcinogenesis or crisis. Furthermore, the TIMELESS-TIPIN complex and CLASPIN may promote replication fork protection independently of CHK1 activation. Normal human fibroblasts (NHF) were depleted of ATR, CHK1, TIMELESS, TIPIN or CLASPIN and chromosomal aberrations, DNA synthesis, activation of the DNA damage response (DDR) and clonogenic survival were evaluated. This work demonstrates in NHF lines from two individuals that ATR and CHK1 promote chromosomal stability by different mechanisms that depletion of CHK1 produces phenotypes that resemble more closely the depletion of TIPIN or CLASPIN than the depletion of ATR, and that TIMELESS has a distinct contribution to suppression of chromosomal instability that is independent of its heterodimeric partner, TIPIN. Therefore, ATR, CHK1, TIMELESS-TIPIN and CLASPIN have functions for preservation of intrinsic chromosomal stability that are separate from their cooperation for activation of the intra-S checkpoint response to experimentally induced replication stress. These data reveal a complex and coordinated program of genome maintenance enforced by proteins known for their intra-S checkpoint function.