Timed inhibition of CDC7 increases CRISPR-Cas9 mediated templated repair.

Repair of double strand DNA breaks (DSBs) can result in gene disruption or gene modification via homology directed repair (HDR) from donor DNA. Altering cellular responses to DSBs may rebalance editing outcomes towards HDR and away from other repair outcomes. Here, we utilize a pooled CRISPR screen to define host cell involvement in HDR between a Cas9 DSB and a plasmid double stranded donor DNA (dsDonor). We find that the Fanconi Anemia (FA) pathway is required for dsDonor HDR and that other genes act to repress HDR. Small molecule inhibition of one of these repressors, CDC7, by XL413 and other inhibitors increases the efficiency of HDR by up to 3.5 fold in many contexts, including primary T cells. XL413 stimulates HDR during a reversible slowing of S-phase that is unexplored for Cas9-induced HDR. We anticipate that XL413 and other such rationally developed inhibitors will be useful tools for gene modification

Histone H4K20 methylation mediated chromatin compaction threshold ensures genome integrity by limiting DNA replication licensing

Cell cycle and replication need to be tightly regulated to ensure genome stability in mammalian cells. Here the authors provide a link between chromatin structure and DNA replication regulation by showing that chromatin compaction limits replication licensing thereby promoting genome integrity

Rif1 S-acylation mediates DNA double-strand break repair at the inner nuclear membrane

Rif1 is involved in telomere homeostasis, DNA replication timing, and DNA double-strand break (DSB) repair pathway choice from yeast to human. The molecular mechanisms that enable Rif1 to fulfill its diverse roles remain to be determined. Here, we demonstrate that Rif1 is S-acylated within its conserved N-terminal domain at cysteine residues C466 and C473 by the DHHC family palmitoyl acyltransferase Pfa4. Rif1 S-acylation facilitates the accumulation of Rif1 at DSBs, the attenuation of DNA end-resection, and DSB repair by non-homologous end-joining (NHEJ). These findings identify S-acylation as a posttranslational modification regulating DNA repair. S-acylated Rif1 mounts a localized DNA-damage response proximal to the inner nuclear membrane, revealing a mechanism of compartmentalized DSB repair pathway choice by sequestration of a fatty acylated repair factor at the inner nuclear membrane

Ortholog of the polymerase theta helicase domain modulates DNA replication in Trypanosoma cruzi

DNA polymerase theta (Polθ), a member of the DNA polymerase family A, exhibits a polymerase C-terminal domain, a central domain, and an N-terminal helicase domain. Polθ plays important roles in DNA repair via its polymerase domain, regulating genome integrity. In addition, in mammals, Polθ modulates origin firing timing and MCM helicase recruitment to chromatin. In contrast, as a model eukaryote, Trypanosoma cruzi exhibits two individual putative orthologs of Polθ in different genomic loci; one ortholog is homologous to the Polθ C-terminal polymerase domain, and the other is homologous to the Polθ helicase domain, called Polθ-polymerase and Polθ-helicase, respectively. A pull-down assay using the T. cruzi component of the prereplication complex Orc1/Cdc6 as bait captured Polθ-helicase from the nuclear extract. Orc1/Cdc6 and Polθ-helicase directly interacted, and Polθ-helicase presented DNA unwinding and ATPase activities. A T. cruzi strain overexpressing the Polθ-helicase domain exhibited a significantly decreased amount of DNA-bound MCM7 and impaired replication origin firing. Taken together, these data suggest that Polθ-helicase modulates DNA replication by directly interacting with Orc1/Cdc6, which reduces the binding of MCM7 to DNA and thereby impairs the firing of replication origins

Measurement of the forward-backward asymmetry in the B→K(*) μ\u3csup\u3e+\u3c/sup\u3eμ\u3csup\u3e-\u3c/sup\u3e decay and first observation of the Bs0→μ\u3csup\u3e+\u3c/sup\u3eμ\u3csup\u3e-\u3c/sup\u3e decay

We reconstruct the rare decays B+→K+μ +μ-, B0→K*(892)0μ +μ-, and Bs0→(1020)μ+μ - in a data sample corresponding to 4.4fb-1 collected in pp̄ collisions at √s=1.96TeV by the CDF II detector at the Tevatron Collider. Using 121±16 B+→K+μ +μ- and 101±12 B0→K*0μ +μ- decays we report the branching ratios. In addition, we report the differential branching ratio and the muon forward-backward asymmetry in the B+ and B0 decay modes, and the K*0 longitudinal polarization fraction in the B0 decay mode with respect to the squared dimuon mass. These are consistent with the predictions, and most recent determinations from other experiments and of comparable accuracy. We also report the first observation of the Bs0→μ+μ- decay and measure its branching ratio BR(Bs0→μ+μ-)= [1.44±0.33±0.46]×10-6 using 27±6 signal events. This is currently the most rare Bs0 decay observed. © 2011 American Physical Society

Search for a new heavy gauge boson W′ with event signature electron+missing transverse energy in pp̅ collisions at √s=1.96 TeV

We present a search for a new heavy charged vector boson W′ decaying to an electron-neutrino pair in pp̅ collisions at a center-of-mass energy of 1.96 TeV. The data were collected with the CDF II detector and correspond to an integrated luminosity of 5.3  fb-1. No significant excess above the standard model expectation is observed and we set upper limits on σ·B(W′→eν). Assuming standard model couplings to fermions and the neutrino from the W′ boson decay to be light, we exclude a W′ boson with mass less than 1.12  TeV/c2 at the 95% confidence level.We thank the Fermilab staff and the technical staffs of the participating institutions for their vital contributions. This work was supported by the U.S. Department of Energy and National Science Foundation; the Italian Istituto Nazionale di Fisica Nucleare; the Ministry of Education, Culture, Sports, Science and Technology of Japan; the Natural Sciences and Engineering Research Council of Canada; the National Science Council of the Republic of China; the Swiss National Science Foundation; the A. P. Sloan Foundation; the Bundesministerium für Balduin Una Forschung, Germany; the World Class University Program, the National Research Foundation of Korea; the Science and Technology Facilities Council and the Royal Society, United Kingdom; the Institut National de Physique Nucleaire et Physique des Particules/CNRS and Universite Pierre et Marie Curie; the Russian Foundation for Basic Research; the Ministerio de Ciencia e Innovación, and Programa Consolider-Ingenio 2010, Spain; the Slovak R&D Agency; and the Academy of Finland

Search for New Dielectron Resonances and Randall-Sundrum Gravitons at the Collider Detector at Fermilab

A search for new dielectron-mass resonances using data recorded by the CDF II detector and corresponding to an integrated luminosity of 5.7fb-1 is presented. No significant excess over the expected standard model prediction is observed. In this data set, an event with the highest dielectron mass ever observed (960GeV/c2) was recorded. The results are interpreted in the Randall-Sundrum (RS) model. Combined with the 5.4fb-1 diphoton analysis, the RS-graviton lower-mass limit for the coupling k/M ̄Pl=0.1 is 1058GeV/c2, making it the strongest limit to date. © 2011 American Physical Society