Broken Ends Research Articles

Precise and Predictable CRISPR Chromosomal Rearrangements Reveal Principles of Cas9-Mediated Nucleotide Insertion

Chromosomal rearrangements including large DNA-fragment inversions, deletions, and duplications by Cas9 with paired sgRNAs are important to investigate genome structural variations and developmental gene regulation, but little is known about the underlying mechanisms. Here, we report that disrupting CtIP or FANCD2, which have roles in alternative non-homologous end joining, enhances precise DNA-fragment deletion. By analyzing the inserted nucleotides at the junctions of DNA-fragment editing of deletions, inversions, and duplications and characterizing the cleaved products, we find that Cas9 endonucleolytically cleaves the noncomplementary strand with a flexible scissile profile upstream of the -3 position of the PAM site invivo and invitro, generating double-strand break ends with 5' overhangs of 1-3 nucleotides. Moreover, we find that engineered Cas9 nucleases have distinct cleavage profiles. Finally, Cas9-mediated nucleotide insertions are nonrandom and are equal to the combined sequences upstream of both PAM sites with predicted frequencies. Thus, precise and predictable DNA-fragment editing could be achieved by perturbing DNA repair genes and using appropriate PAM configurations.

NHEJ Contributes to the Fast Repair of Radiation-induced DNA Double-strand Breaks at Late Prophase I Telomeres.

Exposure of cells to ionizing radiation induces DNA double-strand breaks. To repair double-strand breaks correctly, cells must distinguish between the ends of chromosomes (telomeres) and DNA double-strand breaks within chromosomes. Double-strand breaks in telomeric DNA may lead to telomere shortening and mutagenesis. Eukaryotic cells repair double-strand breaks primarily by two mechanisms: error-free homologous recombination and error-prone nonhomologous end joining, of which homologous recombination is used in early meiotic prophase I to create recombined haploid gametes by two meiotic cell divisions lacking an intervening S-phase. Genotoxic exposures put meiosis at risk to transmit mutations, and ionizing radiation is known to induce large double-strand break-marking phospho (gamma)-H2AX foci along the cores and ends of mouse meiotic chromosomes. However, it remained unclear through which repair pathway the ionizing radiation-induced telomeric double-strand breaks are repaired in late prophase I spermatocytes. Using male wild-type and nonhomologous end joining-deficient (severe combined immunodeficient) mice, this study investigated the kinetics of in vivo double-strand break formation and repair at telomeres of late prophase I chromosomes up to 12 h after 0.5 Gy of whole-body gamma irradiation. Late pachytene and diplotene spermatocytes revealed overlapping gamma-H2AX and telomere repeat signal foci, indicating telomeric DNA damage. The comparison of double-strand break repair rates at telomeres and internal prophase chromosome sites revealed a more rapid double-strand break repair at wild-type telomeres during the first hour after irradiation. Increased double-strand break foci numbers at nonhomologous end joining-deficient telomeres and chromosomes and a slowed repair rate in this DNA-dependent protein kinase catalytic subunit mutant suggest that the fast repair of double-strand breaks in telomeric DNA repeats during late prophase I is largely mediated by canonical nonhomologous end joining.

Seasonal Body Composition Variation Amongst Elite European Professional Soccer Players: An Approach of Talent Identification.

The primary aim of the investigation was to study the seasonal changes in body composition in elite European soccer players and identify key playing positional differences. Twenty-two players (age = 24 ± 3.7 years, body height = 180.45 ± 5.12 cm, body mass = 76.66 ± 5.34 kg) were tested. Players’ mass (kg), lean body mass (LBM), fat free mass (FFM), fat mass (FM), muscle girths (MG) and sum of 8 skinfolds (mm) were measured across 5 time points (T0 = Start of pre-season training; T1 = End of pre-season training; T2 = Mid-season; T3 = End of mid-season break; T4 = End of season). Players were divided into their tactical positional roles for analysis. The specific positions they were divided into included defenders (n = 8), midfielders (n = 8) and forwards (n = 6). Assessment of training and matchplay exposure were also recorded. Sites-4, Sites-7, Sites-8 and Fat Mass decreased dramatically from T0 to T1 and T2 in all playing positions (p < 0.01), while no remarkable differences were found between T2, T3 and T4. Except for defenders, calf girth and lean mass were higher in T2, T3 and T4 compared to T0 and T1 (p < 0.01). Midfielders were found to be leaner than forwards and defenders in all data collection time point sessions. Defenders showed higher values in calf girth and lean body mass than midfielders and forwards. It can be concluded from this investigation that there are large variances n positional body composition profiles amongst professional European soccer players. Furthermore, significant changes are prevalent and occur across the season from LBM, FFM, MG and skinfold assessment amongst European elite level soccer players.

DNA-bound ubiquitin coordinates repair

DNA Repair Ubiquitylation is a posttranslational modification that reversibly alters various protein properties. Liu et al. discovered that Lys63-linked polyubiquitin chains bound to the free ends of double-stranded DNA, bridged the broken ends of DNA, and recruited repair proteins. Ubiquitins with DNA-binding motif mutations were found in several types of tumors. When expressed in cultured cells, these mutant ubiquitins impaired the cellular response to DNA-damaging agents, suggesting that it might be possible to therapeutically exploit these mutations in some cancer patients. Sci. Signal. 11 , eaar8133 (2018).

Probing the evolutionary conserved residues Y204, F259, S400 and W590 that shape the catalytic groove of human TDP1 for 3′- and 5′-phosphodiester-DNA bond cleavage

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is an ubiquitous DNA repair enzyme present in yeast, plants and animals. It removes a broad range of blocking lesions at the ends of DNA breaks. The catalytic core of TDP1 consists in a pair of conserved histidine-lysine-asparagine (HKN) motifs. Analysis of the human TDP1 (hTDP1) crystal structure reveals potential involvement of additional residues that shape the substrate binding site. In this biochemical study, we analyzed four such conserved residues, tyrosine 204 (Y204), phenylalanine 259 (F259), serine 400 (S400) and tryptophan 590 (W590). We show that the F259 residue of hTDP1 is critical for both 3′- and 5′-phosphodiesterase catalysis. We propose that the double π-π interactions of the F259 residue with the −2 and −3 nucleobases serve to position the nucleopeptide substrate in phase with the active site histidines of hTDP1. Mutating Y204 of hTDP1 to phenylalanine (Y204F), as in fly and yeast TDP1 enzymes, had minor impact on TDP1 activity. In constrast, we find that S400 enhances 3′-processing activity while it suppresses 5′-processing activity, thereby promoting specificity for 3′-substrates. W590 is selectively important for 5′-processing. These results reveal the impact of conserved amino acid residues that participate in defining the DNA binding groove around the dual HKN catalytic core motif of TDP1, and their differential roles in facilitating the 3′- vs 5′-end processing activities of hTDP1.

Chromosome ends as adaptive beginnings: the potential role of dysfunctional telomeres in subtelomeric evolvability.

Telomeres serve as protective caps that help the cell differentiate between the naturally occurring ends of chromosomes and double-stranded breaks. When telomere capping function becomes compromised, chromosome ends are subjected to elevated rates of chromosome alterations. These effects can be particularly dramatic in the telomere-adjacent subtelomeric region. While the catastrophic impact of severe telomere dysfunction on genome stability has been well documented, the adaptive telomere failure hypothesis considers an alternative role telomere dysfunction may play in adaptive evolution. This hypothesis suggests that low levels of telomere failure, induced by certain environmental stresses, can lead to elevated subtelomeric recombination. Mutational loss, duplication, or modification of subtelomeric contingency genes could ultimately facilitate adaptation by generating novel mutants better able to survive environmental stress. In this perspective, we discuss recent work that examined mild telomere dysfunction and its role in altering the adaptive potential of subtelomeric genes.

TALEN-Induced Double-Strand Break Repair of CTG Trinucleotide Repeats

Trinucleotide repeat expansions involving CTG/CAG triplets are responsible for several neurodegenerativedisorders, including myotonic dystrophy and Huntington's disease. Because expansions trigger the disease, contracting repeat length could be a possible approach to gene therapy for these disorders. Here, we show that a TALEN-induced double-strand break was very efficient at contracting expanded CTG repeats in yeast. We show that RAD51, POL32, and DNL4 are dispensable for double-strand break repair within CTG repeats, the only required genes being RAD50, SAE2, and RAD52. Resection was totally abolished in the absence of RAD50 on both sides of the break, whereas it was reduced in a sae2Δ mutant on the side of the break containing the longest repeat tract, suggesting that secondary structures at double-strand break ends must be removed by the Mre11-Rad50 complex and Sae2. Following the TALEN double-strand break, single-strand annealing occurred between both sides of the repeat tract, leading to repeat contraction.

Efficient matching technique for laser detection features of cable cutting traces

A lot of line traces on the bearing surface of broken ends which usually present nonlinear morphological features and have strong randomness were left in the crime scene of cable cutting case. In o ...

Osteoperiosteal decortication and extracortical bone-bridging for treatment of atrophic humeral nonunions

Objective To assess the effectiveness of osteoperiosteal decortication and extracortical bone-bridging in the treatment of atrophic humeral nonunions. Methods Nineteen patients with atrophic humeral nonuninon were treated by osteoperiosteal decortication and extracortical bone-bridging between March 2008 and April 2016. They were 12 men and 7 women, aged from 23 to 68 years (mean, 36.6 years). The fracture was located at the left side in 10 cases and at the right side in 9. Before admission to our hospital, 8 had received surgery once, 6 twice and 5 thrice. The time from fracture to hospitalization ranged from 12 to 106 months (average, 26.3 months). Shoulder function was evaluated by Neer scoring and elbow function by Mayo elbow performance score (MEPS) at final follow-ups. Results All incisions healed by first intention. Two cases reported transient radial nerve symptoms of numbness. All the 19 patients were followed up for 28.9 months on average (range, from 13 to 78 months). Radiographic examinations showed signs of bone remodeling, disappearance of fracture lines and formation of extracortical bone bridge at 6 to 8 weeks after operation. All of them achieved radiographic union within 10 to 46 weeks (16.8 weeks on average). The Neer scores averaged 82.5 (range, from 70 to 98), giving 12 excellent cases, 5 good ones and 2 fair ones. The MEPS averaged 84.4 (range, from 70 to 96), giving 11 excellent cases, 5 good ones and 3 fair ones. Conclusion Osteope-riosteal decortication and extracortical bone-bridging in treatment of atrophic humeral nonunions can effectively induce osteogenesis and increase stability of broken ends, promoting bone healing. Key words: Shoulder joint; Elbow joint; Humeral fractures; Fractures, ununited; Bone graft

A tensile study of fancy multicount yarn sections

Purpose No study was published about the tensile properties of different sections of multicount yarn. Hence there was a need to conduct a detailed section-wise tensile study of fancy multicounty yarn. The purpose of this study is to identify the different sections of the same multicount yarn and test them separately. Design/methodology/approach Multicount yarn with slubs were prepared on a ring frame with the Amsler Textile Effect System. Different yarn sections from each multicount yarn are identified as fine normal, fine slub, medium normal, medium slub, coarse normal, coarse slub and the changeover sections. These sections are tested for the breaking load and breaking elongation. Broken ends of the yarn sections are also studied, and these broken ends are classified as sharp broken end, tapered broken end and slipped broken end. A study is also conducted on the location of break or the place of break at the changeover sections. Findings It was found that the twist of yarn at slub sections was lower than the twist at normal sections. In spite of the low yarn twist at slub sections, the breaking load of the yarn was higher at slub sections than at normal sections by 12 to 30 per cent owing to the presence of more number of fibres that share the breaking load. The breaking load at the changeover section (normal to slub and slub to normal) was found to be higher than that at normal section and lower than at slub sections. No significant difference was found between the breaking load of the two changeover sections (normal to slub and slub to normal). The majority of breaks in normal yarn sections were of the tapered broken end type and the majority of breaks in slub yarn sections were slipped broken ends. Originality/value Efforts are taken to identify different sections of the same multicount yarn with the help of specially designed square black board and tensile study of these sections are carried out.

Mapping E. coli Topoisomerase IV Binding and Activity Sites.

This methods article described a protocol aiming at mapping E. coli Topoisomerase IV (Topo IV) binding and cleavage activity sites on the genome. The approach is readily applicable to any Type II topoisomerase on a broad variety of gram-positive and gram-negative bacterial species. Conventional ChIP-seq of flag tagged Topo IV subunits and a novel method aimed at trapping only DNA bound to active Topo IV (called NorfliP) are described. NorfliP relies on the ability of norfloxacin, a quinolone drug, to cross-link the 5' ends of the DNA breaks with the catalytic tyrosine of bacterial Type II topoisomerases. These methods give complementary results and their combination brought important insights onboth the function and regulation of Topo IV.

A curious new role for MRN in Schizosaccharomyces pombe non-homologous end-joining

Chromosomal breaks can be healed by several repair processes, including one called non-homologous end-joining (NHEJ) where the two broken ends are ligated together with a loss of 0-5bp of DNA. The protein requirements for NHEJ of cut DNA ends in the budding yeast Saccharomyces cerevisiae include its version of the Mre11-Rad50-Nbs1 (MRN) complex. In contrast, the fission yeast Schizosaccharomyces pombe and mammalian cells do not require MRN for this process. Recent work in S. pombe used transposon excision to generate breaks that were capped by DNA hairpins, which must be opened to produce ligatable ends. Repair in S. pombe was through an NHEJ reaction that now requires MRN. Surprisingly, wild type cells and MRN mutants that lack nuclease activity showed the same levels of excision. These genetic results suggest that MRN recruits an unknown hairpin-opening nuclease for this unusual NHEJ reaction.

The DNA-Binding Polyamine Moiety in the Vectorized DNA Topoisomerase II Inhibitor F14512 Alters Reparability of the Consequent Enzyme-Linked DNA Double-Strand Breaks.

Poisons of topoisomerase II (TOP2) kill cancer cells by preventing religation of intermediate DNA breaks during the enzymatic process and thus by accumulating enzyme-drug-DNA complexes called TOP2 cleavage-complex (TOP2cc). F14512 is a highly cytotoxic polyamine-vectorized TOP2 inhibitor derived from etoposide and currently in clinical trials. It was shown in vitro that F14512 has acquired DNA-binding properties and that the stability of TOP2cc was strongly increased. Paradoxically, at equitoxic concentrations in cells, F14512 induced less DNA breaks than etoposide. Here, we directly compared etoposide and F14512 for their rates of TOP2cc production and resolution in human cells. We report that targeting of TOP2α and not TOP2β impacts cell killing by F14512, contrary to etoposide that kills cells through targeting both isoforms. Then, we show that despite being more cytotoxic, F14512 is less efficient than etoposide at producing TOP2α cleavage-complex (TOP2αcc) in cells. Finally, we report that compared with TOP2αcc mediated by etoposide, those generated by F14512 persist longer in the genome, are not dependent on TDP2 for cleaning break ends from TOP2α, are channeled to a larger extent to resection-based repair processes relying on CtIP and BRCA1 and promote RAD51 recruitment to damaged chromatin. In addition to the addressing of F14512 to the polyamine transport system, the properties uncovered here would be particularly valuable for a therapeutic usage of this new anticancer compound. More generally, the concept of increasing drug cytotoxicity by switching the repair mode of the induced DNA lesions via addition of a DNA-binding moiety deserves further developments. Mol Cancer Ther; 16(10); 2166-77. ©2017 AACR.

Clinical efficacy of the laparoscope-assisted transanal total mesorectal excision for middle-low rectal cancer

Objective To explore the clinical efficacy of laparoscope-assisted transanal total mesorectal excision (La-TaTME) for middle-low rectal cancer. Methods The retrospective cross-sectional study was conducted. The clinical data of 16 patients with middle-low rectal cancer who underwent La-TaTME in the Peking University Third Hospital from August 2015 to August 2016 were collected. Sequential surgery of La-TaTME was applied to patients in the same team, with laparoscopic surgery first and then transanal surgery. Observation indicators: (1) operation and postoperative recovery situations: conversion to open surgery, anastomosis method, operation time, volume of intraoperative blood loss, intraoperative complications, time for out-of-bed activity, time for liquid diet intake, postoperative complications and duration of postoperative hospital stay. (2) postoperative pathological situations: length of surgical specimen, tumor diameter, distance from tumor to resected distant intestinal canal, complete degree of mesorectum, circumferential resection margin, pathological T stage, pathological N stage, number of lymph node detected and tumor cell differentiation. (3) follow-up. Patients in stage Ⅲ-Ⅳ of TNM stage of RC underwent postoperative adjuvant chemotherapy. Follow-up using outpatient examination was performed once every 3 months postoperatively to detect the patients′ survival and tumor recurrence up to December 2016. Measurement data were represented as M (range). Results (1) Operation and postoperative recovery situations: all the 16 patients underwent successful La-TaTME without conversion to open surgery, including 10 with colorectal anastomosis, 3 with colon-canalis analis anastomosis and 3 with permanent colostomy. Operation time and volume of intraoperative blood loss were 290 minutes (range, 215-420 minutes) and 50 mL (range, 30-100 mL), respectively. One patient had intraoperative complication, showing broken ends ischemia of sigmoid colon after dragging out resected rectum from the anus, following free splenic flexure of colon, about 5 cm ischemic sigmoid colon were resected, and descending colon-rectum anastomosis was performed. Time for out-of-bed activity and time for liquid diet intake were 1 days (range, 1-3 days) and 2 days (range, 1-9 days), respectively. Among 3 patients with postoperative complications (Ⅱ stage of Clavien-Dindo), 2 with incomplete intestinal obstruction were improved by gastrointestinal decompression and total parenteral nutrition, and 1 with presacral infection was improved by drainage and antibiotic therapy. Duration of postoperative hospital stay was 7 days (range, 5-21 days). (2) Postoperative pathological situations: length of surgecal specimen, tumor diameter and distance from tumor to resected distant intestinal canal were respectively 18.0 cm (range, 12.0-24.0 cm), 3.5 cm (range, 0.5-6.8 cm) and 2.5 cm (range, 1.0-5.0 cm). Evaluation of mesorectum of surgical specimen: 14 patients had complete mesorectum of surgical specimen and 2 had nearly complete mesorectum. There was no residual tumor at circumferential resection margin, proximal and distal ends. Pathological T stage of 16 patients: T0 (pathological complete response after neoadjuvant therapy), T1, T2 and T3 stages were found in 1, 1, 4 and 10 patients, respectively. Pathological N stage: 12, 2 and 2 patients were detected in N0, N1 and N2 stages, respectively. Number of lymph node detected was 16 (range, 6-32). Tumor cell differentiation: no tumor cell (pathological complete response after neoadjuvant therapy), high-, moderate- and low-differentiated tumors were respectively detected in 1, 2, 7 and 6 patients. (3) Follow-up. All the patients were followed up for 12 months (range, 4-16 months). There were no local tumor recurrence or distant metastasis and death. Conclusion La-TaTME may be a new, safe and effective resection for middle-low rectal cancer. Key words: Rectal neoplasms, middle-low; Total mesorectal excision; Transanal; Laparoscopy

Recent advances in the study of immunodeficiency and DNA damage response.

DNA breaks can be induced by exogenous stimuli or by endogenous stress, but are also generated during recombination of V, D, and J genes (V(D)J recombination), immunoglobulin class switch recombination (CSR). Among various DNA breaks generated, DNA double strand break (DSB) is the most deleterious one. DNA damage response (DDR) is initiated when DSBs are detected, leading to DNA break repair by non-homologous end joining (NHEJ). The process is critically important for the generation of diversity for foreign antigens; and failure to exert DNA repair leads to immunodeficiency such as severe combined immunodeficiency and hyper-IgM syndrome. In V(D)J recombination, DSBs are induced by RAG1/2; and generated post-cleavage hairpins are resolved by Artemis/DNA-PKcs/KU70/KU80. DDR is initiated by ataxia-telangiectasia mutated as a master regulator together with MRE11/RAD50/NBS1 complex. Finally, DSBs are repaired by NHEJ. The defect of one of the molecules shows various degree of immunodeficiency and radiosensitivity. Upon CSR inducing signal, DSBs induced by activation-induced cytidine deaminase and endonucleases elicit DDR. Broken ends are repaired either by NHEJ or by mismatch repair system. Patients with radiosensitive SCID require hematopoietic cell transplantation as a curative therapy; but the procedures for eradication of recipient hematopoietic cells are often associated with severe toxicity.

Efficient Recreation of t(11;22) EWSR1-FLI1+ in Human Stem Cells Using CRISPR/Cas9

SummaryEfficient methodologies for recreating cancer-associated chromosome translocations are in high demand as tools for investigating how such events initiate cancer. The CRISPR/Cas9 system has been used to reconstruct the genetics of these complex rearrangements at native loci while maintaining the architecture and regulatory elements. However, the CRISPR system remains inefficient in human stem cells. Here, we compared three strategies aimed at enhancing the efficiency of the CRISPR-mediated t(11;22) translocation in human stem cells, including mesenchymal and induced pluripotent stem cells: (1) using end-joining DNA processing factors involved in repair mechanisms, or (2) ssODNs to guide the ligation of the double-strand break ends generated by CRISPR/Cas9; and (3) all-in-one plasmid or ribonucleoprotein complex-based approaches. We report that the generation of targeted t(11;22) is significantly increased by using a combination of ribonucleoprotein complexes and ssODNs. The CRISPR/Cas9-mediated generation of targeted t(11;22) in human stem cells opens up new avenues in modeling Ewing sarcoma.

Vascularized distal radius graft of 1, 2 intercompartmental supraretinacular artery for scaphoid nonunion

Objective To study the techniques and outcomes of using a retrograde pedicled vascularized radius bone flap based on the 1st, 2nd intercompartmental supraretinacular artery (1, 2ICSRA) for scaphoid nonunion through dorsal and palmar approaches. Methods Between March 2013 and April 2015, 16 patients with scaphoid nonunion were treated by surgery through palmar and dorsal approaches. There were 14 males and 2 females with an average age of 29.1 years (range, 19-51 years). The location of lesion was 6 cases on the left and 10 cases on the right. 5 cases were treated with plaster fixation. 11 cases weren’t treated after wrists injured. The preoperative time was 6-26 months, with an average of 16.5 months. The operation was completed within one incision. The bone flaps based on 1, 2ICSRA were taken through dorsal approach. The broken ends of the scaphoid fracture were cleaned, and the deformity was corrected with bone grafting and internal fixation through palmar approach. Time of fracture union and wrist pain resolution was evaluated. Wrist motion and grip strength were measured and compared. The result of the latest follow-up was used as the criterion for efficacy evaluation. The wrist pain was evaluated using visual analogue scale (VAS) and the postoperative wrist function based on the modified Mayo wrist score was recorded. Results Post-operative follow up ranged from 6-19 months, with an average of 11 months. Bone union was achieved in all the cases, so the healing rate was 100%. 7 cases were healed in 11 weeks. 5 cases were healed in 13 weeks. 3 cases were healed in 15 weeks. 1 case was healed in 17 weeks. The average healing time was 12.5 weeks. The humpback deformity of Scaphoid and DISI of 7 cases were corrected. The bending of wrist could reach 60.2°±3.2°, and the elongation could reach 51.3°± 3.5°. The radial deviation of wrist could reach 13.6°±1.42°, and ulnar deviation could reach 24.4°±1.8°. The range of grip strength was 29-64 kg, with an average of 45 kg. The 16 patients returned to normal work. The pain after wrist movement of 14 cases was completely disappeared, and the VAS was 0. Two cases felt slight pain when wrist was overworked, and the VAS were 0.9 and 1.2. The wrist joint function of patients recovered well. The modified Mayo score was 69-99, with an average of 90.75. The functional results were 12 excellent, 2 good, and 2 fair, and the excellent and good rate was 87.5% (14/16) . No complications such as infection, failure of screws, orthopaedic arthritis or scaphoid necrosis were found during the follow-up period. Conclusion The technique of 1, 2 ICSRA pedicled bone graft and palmar bone grafting for nonunion of scaphoid can provide convenience for us to deal with the broken ends of the scaphoid fracture, correct the malformation and graft bone through dorsal and palmar approaches. This method can protect the blood supply of the scaphoid, and promote scaphoid union. Key words: Scaphoid bone; Fractures, ununited; Bone transplantation

Abstract IA20: Phosphorylation of BRCA1 by CHK2 mediates resection activity and recruitment of BRCA2

Abstract There is genetic evidence that Chk2, BRCA1 and BRCA2 are functioning in the same pathway of DNA repair, but the links between the proteins are poorly understood. We have previously shown that BRCA1's regulation of homologous recombination (HR) is dependent on Chk2-mediated phosphorylation of BRCA1 at serine 988 (BRCA1-S988). We also found that BRCA1 regulates BRCA2 recruitment in response to DNA damage, which is also dependent on the phosphorylation of BRCA1 at Serine-988. Furthermore, in HCT116/Chk2-/- cells and in MCF7 cells with depleted Chk2, the same effects on HR and BRCA2 recruitment has been found as found in cells expressing the BRCA1-S988A protein. However, in both cases, BRCA1 is recruited to chromatin and into nuclear foci, so the effect of Chk2 is not affecting the recruitment of BRCA1. BRCA1-S988A shows a defect in RPA phosphorylation in response to replication stress and DNA damage. After global DNA damage with X-rays, MRE11 foci form at double-strand breaks, and in S and G2 phases of the cell cycle, an association with CTIP is observed transiently. In the presence of BRCA1-S988A, MRE11 co-localized with CTIP persists for much longer, and results in reduced single-stranded DNA bound by RPA. In response to a site-specific DSB induced by I-SceI nuclease, BRCA1-S988A can localize to the vicinity of the break, but not directly at the broken end, as observed by chromatin immuno-precipitation. A physical assay of resection, which distinguishes single-stranded DNA from double-stranded DNA by its susceptibility to restriction enzyme digestion, shows that BRCA1-S988A expressing cells have an initiation of resection, but a failure to extend resection up to 2 kb observed in wild-type cells. The recruitment of Exo1 is being investigated. Therefore, these results demonstrate that BRCA1-S988 plays key role in regulating the extent of resection, which in turn impedes the recruitment of BRCA2 to sites of resected DSB. Chk2 inhibition may exacerbate the defect in HR, particularly in cells with a partial or mild HR-deficiency, which is found quite commonly in human tumor cells, suggesting that combination of Chk2 and PARP inhibitors may be a useful combination DNA repair based therapy. Citation Format: Simon N. Powell. Phosphorylation of BRCA1 by CHK2 mediates resection activity and recruitment of BRCA2 [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr IA20.

The effect of physical activity on sleep quality, well-being, and affect in academic stress periods.

The stress-buffering hypothesis postulates that physical activity and exercise can buffer the negative effects of (academic) stress on health. It still remains an open question whether students, who regularly engage in physical activity and exercise within their academic examination period, can successfully diminish these negative effects. Sixty-four subjects participated in this study and completed a total of five surveys, with T1 at the end of the semester break (baseline) and T2–T5 being presented every Friday in the last 4 weeks of the semester (examination period). They were asked to answer questions about their activity level, sleep quality, well-being and affect. Hierarchical linear models showed significant dependencies on time for all dependent measures. The expansion of the model for exercise also showed significant main effects of this predictor on well-being and positive affect (PA) and negative affect. Moreover, significant interactions with time for sleep quality and PA were found. Results suggest that physical activity and exercise in the academic examination period may be able to buffer the negative effects of stress on health-related outcomes. Therefore, activity levels should be maintained in times of high stress to prevent negative effects on sleep, well-being and affect in students.

Inhibition of DNA2 nuclease as a therapeutic strategy targeting replication stress in cancer cells

Replication stress is a characteristic feature of cancer cells, which is resulted from sustained proliferative signaling induced by activation of oncogenes or loss of tumor suppressors. In cancer cells, oncogene-induced replication stress manifests as replication-associated lesions, predominantly double-strand DNA breaks (DSBs). An essential mechanism utilized by cells to repair replication-associated DSBs is homologous recombination (HR). In order to overcome replication stress and survive, cancer cells often require enhanced HR repair capacity. Therefore, the key link between HR repair and cellular tolerance to replication-associated DSBs provides us with a mechanistic rationale for exploiting synthetic lethality between HR repair inhibition and replication stress. DNA2 nuclease is an evolutionarily conserved essential enzyme in replication and HR repair. Here we demonstrate that DNA2 is overexpressed in pancreatic cancers, one of the deadliest and more aggressive forms of human cancers, where mutations in the KRAS are present in 90–95% of cases. In addition, depletion of DNA2 significantly reduces pancreatic cancer cell survival and xenograft tumor growth, suggesting the therapeutic potential of DNA2 inhibition. Finally, we develop a robust high-throughput biochemistry assay to screen for inhibitors of the DNA2 nuclease activity. The top inhibitors were shown to be efficacious against both yeast Dna2 and human DNA2. Treatment of cancer cells with DNA2 inhibitors recapitulates phenotypes observed upon DNA2 depletion, including decreased DNA double strand break end resection and attenuation of HR repair. Similar to genetic ablation of DNA2, chemical inhibition of DNA2 selectively attenuates the growth of various cancer cells with oncogene-induced replication stress. Taken together, our findings open a new avenue to develop a new class of anticancer drugs by targeting druggable nuclease DNA2. We propose DNA2 inhibition as new strategy in cancer therapy by targeting replication stress, a molecular property of cancer cells that is acquired as a result of oncogene activation instead of targeting currently undruggable oncoprotein itself such as KRAS.