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

Fabrication and verification of conjugated AuNP-antibody nanoprobe for sensitivity improvement in electrochemical biosensors

Abstract This study was designed to obtain covalently coupled conjugates as means for achieving higher stability and better coverage of the AuNPs by antibodies on the particle surface suitable for sensor performance enhancement. Starting by using a modified protocol, colloid gold solution, with mean AuNP core size of ~6 nm was synthesized. The protocol used for conjugation of AuNPs to osteocalcin antibody in this study relies on covalent and electrostatic attractions between constituents. Varieties of conjugates with varying combinations of crosslinkers and different concentrations were successfully synthesized. The obtained products were characterized and their properties were studied to determine the best candidate in sense of antibody - antigen reactivity. Using AuNP-GSH-NHS-Ab combination (1:1:1), the tertiary structure of the protein was maintained and thus the antibody remained functional in the future steps. This one-pot method provided a simple method for covalently coupling antibodies on the particle surface while keeping their functionality intact. The AuNP content of the solution also accelerated electron transfer rate and thus amplifies the detection signal. With the developed and discussed technique herein, a simple solution is modeled to be used for measuring serum levels of biomarkers in single and/or multiplexed sensor systems

Defective Resection at DNA Double-Strand Breaks Leads to De Novo Telomere Formation and Enhances Gene Targeting

The formation of single-stranded DNA (ssDNA) at double-strand break (DSB) ends is essential in repair by homologous recombination and is mediated by DNA helicases and nucleases. Here we estimated the length of ssDNA generated during DSB repair and analyzed the consequences of elimination of processive resection pathways mediated by Sgs1 helicase and Exo1 nuclease on DSB repair fidelity. In wild-type cells during allelic gene conversion, an average of 2–4 kb of ssDNA accumulates at each side of the break. Longer ssDNA is formed during ectopic recombination or break-induced replication (BIR), reflecting much slower repair kinetics. This relatively extensive resection may help determine sequences involved in homology search and prevent recombination within short DNA repeats next to the break. In sgs1Δ exo1Δ mutants that form only very short ssDNA, allelic gene conversion decreases 5-fold and DSBs are repaired by BIR or de novo telomere formation resulting in loss of heterozygosity. The absence of the telomerase inhibitor, PIF1, increases de novo telomere pathway usage to about 50%. Accumulation of Cdc13, a protein recruiting telomerase, at the break site increases in sgs1Δ exo1Δ, and the requirement of the Ku complex for new telomere formation is partially bypassed. In contrast to this decreased and alternative DSB repair, the efficiency and accuracy of gene targeting increases dramatically in sgs1Δ exo1Δ cells, suggesting that transformed DNA is very stable in these mutants. Altogether these data establish a new role for processive resection in the fidelity of DSB repair

The Transcriptional Response to DNA-Double-Strand Breaks in Physcomitrella patens

The model bryophyte Physcomitrella patens is unique among plants in supporting the generation of mutant alleles by facile homologous recombination-mediated gene targeting (GT). Reasoning that targeted transgene integration occurs through the capture of transforming DNA by the homology-dependent pathway for DNA double-strand break (DNA-DSB) repair, we analysed the genome-wide transcriptomic response to bleomycin-induced DNA damage and generated mutants in candidate DNA repair genes. Massively parallel (Illumina) cDNA sequencing identified potential participants in gene targeting. Transcripts encoding DNA repair proteins active in multiple repair pathways were significantly up-regulated. These included Rad51, CtIP, DNA ligase 1, Replication protein A and ATR in homology-dependent repair, Xrcc4, DNA ligase 4, Ku70 and Ku80 in non-homologous end-joining and Rad1, Tebichi/polymerase theta, PARP in microhomology-mediated end-joining. Differentially regulated cell-cycle components included up-regulated Rad9 and Hus1 DNA-damage-related checkpoint proteins and down-regulated D-type cyclins and B-type CDKs, commensurate with the imposition of a checkpoint at G2 of the cell cycle characteristic of homology-dependent DNA-DSB repair. Candidate genes, including ATP-dependent chromatin remodelling helicases associated with repair and recombination, were knocked out and analysed for growth defects, hypersensitivity to DNA damage and reduced GT efficiency. Targeted knockout of PpCtIP, a cell-cycle activated mediator of homology-dependent DSB resection, resulted in bleomycin-hypersensitivity and greatly reduced GT efficiency

Effects of Preoperative Aspirin Use on Mortality in Coronary Artery Bypass Grafting Patients

Background and Aim: Discontinuing aspirin use in patients before coronary artery bypass grafting (CABG) has focused on bleeding risks. The aim of this study was to determind the effect of aspirin use on overall mortality with this procedure.Materials and Methods: In a retrospective review was under taken of 2,252 consecutive isolated coronary artery bypass grafting (CABG) procedures performed between July 1995 and May 3003 in Tehran, Shriati hospital. Patients who had isolated CABG operations and received aspirin were analyzed and compared to nonusers undergoing similar bypass procedures during the same period. Aspirin use was defined by identification of ingestion within 7 days before the operation.Results: CABG patients using preoperative aspirin were less likely to experience in hospital hemorrhage compared to nonusers (P=0.0001). Significant difference was seen for transfusion of blood products, and need for reexploration for hemorrhage between patients who did and did not receive aspirin (P=0.0001).Mortality in multivariate (odds ratio [OR] =0.12, 95%confidence interval [0.05, 0.28] analysis was less to patients using aspirin compared to nonusers (p=0.0001).Conclusions : Preoperative aspirin use appears to be associated with a decreased risk of mortality in CABG patients with significant increase in hemorrhage, blood product requirements, or related morbidities

Prevalence and Risk Factors of Gastroesophageal Reflux Disease in Tabriz, Iran

"nBackground: Gastroesophageal reflux disease (GERD) is one of the most common gastrointestinal problems in the west while different reports indicate an increase in the prevalence in Iran. The aim of this study was to estimate the preva­lence and clinical spectrum of GERD in staff of a referral hospital and evaluate the risk factors."nMethods: This cross-sectional study using a modified Mayo clinic questionnaire was performed on staff of Imam Hospi­tal, Tabriz, Iran on a pilot of 50 subjects, and a randomly selected group consisted of 522 subjects in the year 2005. GERD symptoms were defined as at least weekly heartburn and/or acid regurgitation during the past year."nResults: Response rate was 95%. Mean age of responders was 40.02±10.72 yr. The prevalence of recurrent heart­burn and/or acid regurgitation experienced at least weekly and monthly was 26.8% and 34.1%, respectively. They were not related to age and gender. The severity of symptoms was mainly reported of a mild to moderate degree. 45% of the cases reported at least one of the atypical symptoms. There was no relation between marriage status and preva­lence of GERD. On the other hand, GERD was less common among cases with no family history of upper gas­trointestinal disease. The prevalence of frequent GERD was more common among medical staff. Increased BMI (but no recent weight gain or lose) was associated with higher prevalence of GERD symptoms only in women. In­terestingly 33% of our population had a history of using antacid or PPIs which was more among cases with frequent GERD symptoms."nConclusion: This study revealed a high prevalence of frequent GERD symptoms in a selected population of Tabriz. Atypi­cal symptoms should be considered in this area. &nbsp