Variation in DNA repair genes XRCC3, XRCC4, XRCC5 and susceptibility to myeloma

Cytogenetic analysis in myeloma reveals marked chromosomal instability. Both widespread genomic alterations and evidence of aberrant class switch recombination, the physiological process that regulates maturation of the antibody response, implicate the DNA repair pathway in disease pathogenesis. We therefore assessed 27 SNPs in three genes (XRCC3, XRCC4 and XRCC5) central to DNA repair in patients with myeloma and controls from the EpiLymph study and from an Irish hospital registry (n = 306 cases, 263 controls). For the haplotype-tagging SNP (htSNP) rs963248 in XRCC4, Allele A was significantly more frequent in cases than in controls (86.4 versus 80.8%; odds ratio 1.51; 95% confidence interval 1.10-2.08; P = 0.0133), as was the AA genotype (74 versus 65%) (P = 0.026). Haplotype analysis was performed using Unphased for rs963248 in combination with additional SNPs in XRCC4. The strongest evidence of association came from the A-T haplotype from rs963248-rs2891980 (P = 0.008). For XRCC5, the genotype GG from rs1051685 was detected in 10 cases from different national populations but in only one control (P = 0.015). This SNP is located in the 3'-UTR of XRCC5. Overall, these data provide support for the hypothesis that common variation in the genes encoding DNA repair proteins contributes to susceptibility to myeloma.

The human coronavirus 229E superfamily 1 helicase has RNA and DNA duplex-unwinding activities with 5'-to-3' polarity.

The human coronavirus 229E replicase gene encodes a protein, p66HEL, that contains a putative zinc finger structure linked to a putative superfamily (SF) 1 helicase. A histidine-tagged form of this protein, HEL, was expressed using baculovirus vectors in insect cells. The purified recombinant protein had in vitro ATPase activity that was strongly stimulated by poly(U), poly(dT), poly(C), and poly(dA), but not by poly(G). The recombinant protein also had both RNA and DNA duplex-unwinding activities with 5'-to-3' polarity. The DNA helicase activity of the enzyme preferentially unwound 5'-oligopyrimidine-tailed, partial-duplex substrates and required a tail length of at least 10 nucleotides for effective unwinding. The combined data suggest that the coronaviral SF1 helicase functionally differs from the previously characterized RNA virus SF2 helicases.

Biochemical characterization of the equine arteritis virus helicase suggests a close functional relationship between arterivirus and coronavirus helicases.

The arterivirus equine arteritis virus nonstructural protein 10 (nsp10) has previously been predicted to contain a Zn finger structure linked to a superfamily 1 (SF1) helicase domain. A recombinant form of nsp10, MBP-nsp10, was produced in Escherichia coli as a fusion protein with the maltose-binding protein. The protein was partially purified by affinity chromatography and shown to have ATPase activity that was strongly stimulated by poly(dT), poly(U), and poly(dA) but not by poly(G). The protein also had both RNA and DNA duplex-unwinding activities that required the presence of 5' single-stranded regions on the partial-duplex substrates, indicating a 5'-to-3' polarity in the unwinding reaction. Results of this study suggest a close functional relationship between the arterivirus nsp10 and the coronavirus helicase, for which NTPase and duplex-unwinding activities were recently demonstrated. In a number of biochemical properties, both arterivirus and coronavirus SF1 helicases differ significantly from the previously characterized RNA virus SF1 and SF2 enzymes. Thus, the combined data strongly support the idea that nidovirus helicases may represent a separate group of RNA virus-encoded helicases with distinct properties.

Single-stranded DNA-binding protein hSSB1 is critical for genomic stability

Single-strand DNA (ssDNA)-binding proteins (SSBs) are ubiquitous and essential for a wide variety of DNA metabolic processes, including DNA replication, recombination, DNA damage detection and repair. SSBs have multiple roles in binding and sequestering ssDNA, detecting DNA damage, stimulating nucleases, helicases and strand-exchange proteins, activating transcription and mediating protein-protein interactions. In eukaryotes, the major SSB, replication protein A (RPA), is a heterotrimer. Here we describe a second human SSB (hSSB1), with a domain organization closer to the archaeal SSB than to RPA. Ataxia telangiectasia mutated (ATM) kinase phosphorylates hSSB1 in response to DNA double-strand breaks (DSBs). This phosphorylation event is required for DNA damage-induced stabilization of hSSB1. Upon induction of DNA damage, hSSB1 accumulates in the nucleus and forms distinct foci independent of cell-cycle phase. These foci co-localize with other known repair proteins. In contrast to RPA, hSSB1 does not localize to replication foci in S-phase cells and hSSB1 deficiency does not influence S-phase progression. Depletion of hSSB1 abrogates the cellular response to DSBs, including activation of ATM and phosphorylation of ATM targets after ionizing radiation. Cells deficient in hSSB1 exhibit increased radiosensitivity, defective checkpoint activation and enhanced genomic instability coupled with a diminished capacity for DNA repair. These findings establish that hSSB1 influences diverse endpoints in the cellular DNA damage response.