DNA resection in eukaryotes: deciding how to fix the break

DNA double-strand breaks are repaired by different mechanisms, including homologous recombination and nonhomologous end-joining. DNA-end resection, the first step in recombination, is a key step that contributes to the choice of DSB repair. Resection, an evolutionarily conserved process that generates single-stranded DNA, is linked to checkpoint activation and is critical for survival. Failure to regulate and execute this process results in defective recombination and can contribute to human disease. Here, I review recent findings on the mechanisms of resection in eukaryotes, from yeast to vertebrates, provide insights into the regulatory strategies that control it, and highlight the consequences of both its impairment and its deregulation

CtIP Mutations Cause Seckel and Jawad Syndromes

Seckel syndrome is a recessively inherited dwarfism disorder characterized by microcephaly and a unique head profile. Genetically, it constitutes a heterogeneous condition, with several loci mapped (SCKL1-5) but only three disease genes identified: the ATR, CENPJ, and CEP152 genes that control cellular responses to DNA damage. We previously mapped a Seckel syndrome locus to chromosome 18p11.31-q11.2 (SCKL2). Here, we report two mutations in the CtIP (RBBP8) gene within this locus that result in expression of C-terminally truncated forms of CtIP. We propose that these mutations are the molecular cause of the disease observed in the previously described SCKL2 family and in an additional unrelated family diagnosed with a similar form of congenital microcephaly termed Jawad syndrome. While an exonic frameshift mutation was found in the Jawad family, the SCKL2 family carries a splicing mutation that yields a dominant-negative form of CtIP. Further characterization of cell lines derived from the SCKL2 family revealed defective DNA damage induced formation of single-stranded DNA, a critical co-factor for ATR activation. Accordingly, SCKL2 cells present a lowered apoptopic threshold and hypersensitivity to DNA damage. Notably, over-expression of a comparable truncated CtIP variant in non-Seckel cells recapitulates SCKL2 cellular phenotypes in a dose-dependent manner. This work thus identifies CtIP as a disease gene for Seckel and Jawad syndromes and defines a new type of genetic disease mechanism in which a dominant negative mutation yields a recessively inherited disorder

CtIP tetramer assembly is required for DNA-end resection and repair.

Mammalian CtIP protein has major roles in DNA double-strand break (DSB) repair. Although it is well established that CtIP promotes DNA-end resection in preparation for homology-dependent DSB repair, the molecular basis for this function has remained unknown. Here we show by biophysical and X-ray crystallographic analyses that the N-terminal domain of human CtIP exists as a stable homotetramer. Tetramerization results from interlocking interactions between the N-terminal extensions of CtIP's coiled-coil region, which lead to a 'dimer-of-dimers' architecture. Through interrogation of the CtIP structure, we identify a point mutation that abolishes tetramerization of the N-terminal domain while preserving dimerization in vitro. Notably, we establish that this mutation abrogates CtIP oligomer assembly in cells, thus leading to strong defects in DNA-end resection and gene conversion. These findings indicate that the CtIP tetramer architecture described here is essential for effective DSB repair by homologous recombination.We thank M. Kilkenny for help with the collection of X-ray diffraction data, A. Sharff and P. Keller for help with X-ray data processing and J.D. Maman for assistance with SEC-MALS. This work was supported by a Wellcome Trust Senior Research Fellowship award in basic biomedical sciences (L.P.), an Isaac Newton Trust research grant (L.P. and O.R.D.) and a Cambridge Overseas Trust PhD studentship (M.D.S.). Research in the laboratory of S.P.J. is funded by Cancer Research UK (CRUK; programme grant C6/A11224), the European Research Council and the European Community Seventh Framework Programme (grant agreement no. HEALTH-F2-2010-259893 (DDResponse)). Core funding is provided by Cancer Research UK (C6946/A14492) and the Wellcome Trust (WT092096). S.P.J. receives his salary from the University of Cambridge, supplemented by CRUK. J.V.F. is funded by Cancer Research UK programme grant C6/A11224 and the Ataxia Telangiectasia Society. R.B. and J.C. are funded by Cancer Research UK programme grant C6/A11224. Y.G. and M.D. are funded by the European Research Council grant DDREAM.This is the accepted manuscript of a paper published in Nature Structural & Molecular Biology, 22, 150–157 (2015) doi: 10.1038/nsmb.293

Relationship of metabolic syndrome and its components with -844 G/A and HindIII C/G PAI-1 gene polymorphisms in Mexican children

<p>Abstract</p> <p>Background</p> <p>Several association studies have shown that -844 G/A and <it>HindIII </it>C/G <it>PAI-1 </it>polymorphisms are related with increase of PAI-1 levels, obesity, insulin resistance, glucose intolerance, hypertension and dyslipidemia, which are components of metabolic syndrome. The aim of this study was to analyze the allele and genotype frequencies of these polymorphisms in <it>PAI-1 </it>gene and its association with metabolic syndrome and its components in a sample of Mexican mestizo children.</p> <p>Methods</p> <p>This study included 100 children with an age range between 6-11 years divided in two groups: a) 48 children diagnosed with metabolic syndrome and b) 52 children metabolically healthy without any clinical and biochemical alteration. Metabolic syndrome was defined as the presence of three or more of the following criteria: fasting glucose levels ≥ 100 mg/dL, triglycerides ≥ 150 mg/dL, HDL-cholesterol < 40 mg/dL, obesity BMI ≥ 95^thpercentile, systolic blood pressure (SBP) and diastolic blood pressure (DBP) ≥ 95^thpercentile and insulin resistance HOMA-IR ≥ 2.4. The -844 G/A and <it>HindIII </it>C/G <it>PAI-1 </it>polymorphisms were analyzed by PCR-RFLP.</p> <p>Results</p> <p>For the -844 G/A polymorphism, the G/A genotype (OR = 2.79; 95% CI, 1.11-7.08; <it>p </it>= 0.015) and the A allele (OR = 2.2; 95% CI, 1.10-4.43; <it>p </it>= 0.015) were associated with metabolic syndrome. The -844 G/A and A/A genotypes were associated with increase in plasma triglycerides levels (OR = 2.6; 95% CI, 1.16 to 6.04; <it>p </it>= 0.02), decrease in plasma HDL-cholesterol levels (OR = 2.4; 95% CI, 1.06 to 5.42; <it>p </it>= 0.03) and obesity (OR = 2.6; 95% CI, 1.17-5.92; <it>p </it>= 0.01). The C/G and G/G genotypes of the <it>HindIII </it>C/G polymorphism contributed to a significant increase in plasma total cholesterol levels (179 vs. 165 mg/dL; <it>p </it>= 0.02) in comparison with C/C genotype.</p> <p>Conclusions</p> <p>The -844 G/A <it>PAI-1 </it>polymorphism is related with the risk of developing metabolic syndrome, obesity and atherogenic dyslipidemia, and the <it>HindIII </it>C/G <it>PAI-1 </it>polymorphism was associated with the increase of total cholesterol levels in Mexican children.</p

Collaborative Action of Brca1 and CtIP in Elimination of Covalent Modifications from Double-Strand Breaks to Facilitate Subsequent Break Repair

Topoisomerase inhibitors such as camptothecin and etoposide are used as anti-cancer drugs and induce double-strand breaks (DSBs) in genomic DNA in cycling cells. These DSBs are often covalently bound with polypeptides at the 3′ and 5′ ends. Such modifications must be eliminated before DSB repair can take place, but it remains elusive which nucleases are involved in this process. Previous studies show that CtIP plays a critical role in the generation of 3′ single-strand overhang at “clean” DSBs, thus initiating homologous recombination (HR)–dependent DSB repair. To analyze the function of CtIP in detail, we conditionally disrupted the CtIP gene in the chicken DT40 cell line. We found that CtIP is essential for cellular proliferation as well as for the formation of 3′ single-strand overhang, similar to what is observed in DT40 cells deficient in the Mre11/Rad50/Nbs1 complex. We also generated DT40 cell line harboring CtIP with an alanine substitution at residue Ser332, which is required for interaction with BRCA1. Although the resulting CtIPS332A/−/− cells exhibited accumulation of RPA and Rad51 upon DNA damage, and were proficient in HR, they showed a marked hypersensitivity to camptothecin and etoposide in comparison with CtIP+/−/− cells. Finally, CtIPS332A/−/−BRCA1−/− and CtIP+/−/−BRCA1−/− showed similar sensitivities to these reagents. Taken together, our data indicate that, in addition to its function in HR, CtIP plays a role in cellular tolerance to topoisomerase inhibitors. We propose that the BRCA1-CtIP complex plays a role in the nuclease-mediated elimination of oligonucleotides covalently bound to polypeptides from DSBs, thereby facilitating subsequent DSB repair

Hypertension in acute ischemic stroke - A compensatory mechanism or an additional damaging factor?

BACKGROUND: In acute ischemic stroke, a transient blood pressure (BP) elevation is common, but the best management is still unknown. Therefore, we investigated retrospectively the relationship between BP after ischemic stroke and neurological outcome (evaluated by means of the National Institutes of Health Stroke Scale score at day 7). METHODS: The medical records of 92 consecutive patients with acute ischemic stroke, aged 47 to 96 years, were examined. Blood pressure was measured on admission, 4 times during the first 24 hours, 3 times daily for the first 4 days, and twice daily on day 7 (or at discharge). Antihypertensive treatment was given according to American Heart Association guidelines. RESULTS: The region damaged by the stroke was total anterior in 16 patients (17%), partial anterior in 30 (33%), lacunar in 34 (37%), and posterior circulation in 12 (13%). Stroke pathogenesis was cardioembolic in 28 (30%), atherothrombotic in 29 (32%), and lacunar in 34 (37%). The systolic BP range was 140 to 220 mm Hg; diastolic BP, 70 to 110 mm Hg. Initial BP was higher in the group with lacunar infarction than in the other groups (P<.05). The patients with the best outcome had the highest BP during the first 24 hours. The neurological outcome was strongly influenced by baseline stroke severity (NIH Scale score) and admission BP. Better initial neurological conditions and higher initial BP resulted in better neurological outcomes. CONCLUSIONS: The outcome of stroke is influenced by the type of stroke and initial BP. Lacunar stroke and the highest BP on admission carry the best prognosis, whereas the reverse is true for posterior circulation infarction and low BP. We found no evidence that, within the present BP range, hypertension is harmful and that its lowering is beneficial