npg DNA double-strand breaks (DSBs) are critical lesions that can result in cell death or a wide variety of genetic alterations including large-or small-scale deletions, loss of heterozygosity, translocations, and chromosome loss. DSBs are repaired by non-homologous end-joining (NHEJ) and homologous recombination (HR), and defects in these pathways cause genome instability and promote tumorigenesis. DSBs arise from endogenous sources including reactive oxygen species generated during cellular metabolism, collapsed replication forks, and nucleases, and from exogenous sources including ionizing radiation and chemicals that directly or indirectly damage DNA and are commonly used in cancer therapy. The DSB repair pathways appear to compete for DSBs, but the balance between them differs widely among species, between different cell types of a single species, and during different cell cycle phases of a single cell type. Here we review the regulatory factors that regulate DSB repair by NHEJ and HR in yeast and higher eukaryotes. These factors include regulated expression and phosphorylation of repair proteins, chromatin modulation of repair factor accessibility, and the availability of homologous repair templates. While most DSB repair proteins appear to function exclusively in NHEJ or HR, a number of proteins influence both pathways, including the MRE11/RAD50/NBS1(XRS2) complex, BRCA1, histone H2AX, PARP-1, RAD18, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and ATM. DNA-PKcs plays a role in mammalian NHEJ, but it also influences HR through a complex regulatory network that may involve crosstalk with ATM, and the regulation of at least 12 proteins involved in HR that are phosphorylated by DNA-PKcs and/or ATM.

Given its significant role in the maintenance of genomic stability, histone methylation has been postulated to regulate DNA repair. Histone methylation mediates localization of 53BP1 to a DNA double-strand break (DSB) during homologous recombination repair, but a role in DSB repair by nonhomologous end-joining (NHEJ) has not been defined. By screening for histone methylation after DSB induction by ionizing radiation we found that generation of dimethyl histone H3 lysine 36 (H3K36me2) was the major event.Using a novel human cell system that rapidly generates a single defined DSB in the vast majority of cells, we found that the DNA repair protein Metnase (also SETMAR), which has a SET histone methylase domain, localized to an induced DSB and directly mediated the formation of H3K36me2 near the induced DSB. This dimethylation of H3K36 improved the association of early DNA repair components, including NBS1 and Ku70, with the induced DSB, and enhanced DSB repair. In addition, expression of JHDM1a (an H3K36me2 demethylase) or histone H3 in which K36 was mutated to A36 or R36 to prevent H3K36me2 formation decreased the association of early NHEJ repair components with an induced DSB and decreased DSB repair. Thus, these experiments define a histone methylation event that enhances DNA DSB repair by NHEJ.double-strand break | I-Sce-I | chromatin immunoprecipitation | MRN complex | mathematical modeling H istone methylation is highly regulated by a family of proteins termed histone methylases, which usually share a SET domain (1-3). Histone methylation plays a key role in chromatin remodeling and as such regulates transcription, replication, cell differentiation, genome stability, and apoptosis (1-3). Because of its role in replication and genome stability, histone methylation has been hypothesized to play an important role in DNA repair. DNA double-strand breaks (DSBs) are a cytotoxic form of DNA damage that disrupts many of the cellular functions regulated by histone methylation described above (4-6). Previous reports indicate that histone methylation may be important in DNA DSB repair by homologous recombination: The DSB repair component 53BP1, which is required for proper homologous recombination, is recruited to sites of damage by methylated histone H3 lysine 79 (H3K79) and histone H4 lysine 20 (H4K20) (7-9). However, neither H3K79 nor H4K20 methylation is induced by DNA damage (9), so other histone methylation events at sites of DNA damage have been sought. In addition, a mechanism by which histone methylation might regulate NHEJ DSB repair has yet to be defined. In this study, a survey of histone methylation events after DSB induction revealed that the major immediate H3 methylation event is H3K36me2.Metnase is a DNA DSB repair component that is a fusion of a SET histone methylase domain with a nuclease domain and a domain from a member of the transposase/integrase family (10-14). We showed previously that Metnase enhances nonhomologous end-joining (NHEJ) repair of, and survival after, DNA DSBs, and that its SET dom...

DNA double-strand breaks (DSBs) are repaired by nonhomologous end-joining (NHEJ) and homologous recombination (HR). The NHEJ/HR decision is under complex regulation and involves DNA-dependent protein kinase (DNA-PKcs). HR is elevated in DNA-PKcs null cells, but suppressed by DNA-PKcs kinase inhibitors, suggesting that kinase-inactive DNA-PKcs (DNA-PKcs-KR) would suppress HR. Here we use a direct repeat assay to monitor HR repair of DSBs induced by I-SceI nuclease. Surprisingly, DSB-induced HR in DNA-PKcs-KR cells was 2-to 3-fold above the elevated HR level of DNA-PKcs null cells, and ∼4-to 7-fold above cells expressing wild-type DNA-PKcs. The hyperrecombination in DNA-PKcs-KR cells compared to DNA-PKcs null cells was also apparent as increased resistance to DNA crosslinks induced by mitomycin C. ATM phosphorylates many HR proteins, and ATM is expressed at a low level in cells lacking DNA-PKcs, but restored to wild-type level in cells expressing DNA-PKcs-KR. Several clusters of phosphorylation sites in DNA-PKcs, including the T2609 cluster, which is phosphorylated by DNA-PKcs and ATM, regulate access of repair factors to broken ends. Our results indicate that ATM-dependent phosphorylation of DNA-PKcs-KR contributes to the hyperrecombination phenotype. Interestingly, DNA-PKcs null cells showed more persistent ionizing radiation-induced RAD51 foci (but lower HR levels) compared to DNA-PKcs-KR cells, consistent with HR completion requiring RAD51 turnover. ATM may promote RAD51 turnover, suggesting a second (not mutually exclusive) mechanism by which restored ATM contributes to hyperrecombination in DNA-PKcs-KR cells. We propose a model in which DNA-PKcs and ATM coordinately regulate DSB repair by NHEJ and HR.

Metnase is a human protein with methylase (SET) and nuclease domains that is widely expressed, especially in proliferating tissues. Metnase promotes non-homologous end-joining (NHEJ), and knockdown causes mild hypersensitivity to ionizing radiation. Metnase also promotes plasmid and viral DNA integration, and topoisomerase IIα (TopoIIα)-dependent chromosome decatenation. NHEJ factors have been implicated in the replication stress response, and TopoIIα has been proposed to relax positive supercoils in front of replication forks. Here we show that Metnase promotes cell proliferation, but it does not alter cell cycle distributions, or replication fork progression. However, Metnase knockdown sensitizes cells to replication stress and confers a marked defect in restart of stalled replication forks. Metnase promotes resolution of phosphorylated histone H2AX, a marker of DNA double-strand breaks at collapsed forks, and it co-immunoprecipitates with PCNA and RAD9, a member of the PCNA-like RAD9–HUS1–RAD1 intra-S checkpoint complex. Metnase also promotes TopoIIα-mediated relaxation of positively supercoiled DNA. Metnase is not required for RAD51 focus formation after replication stress, but Metnase knockdown cells show increased RAD51 foci in the presence or absence of replication stress. These results establish Metnase as a key factor that promotes restart of stalled replication forks, and implicate Metnase in the repair of collapsed forks.

Background: Vacuolar ATPase (V-ATPase) proton pumps maintain pH homeostasis. Results: We discovered new V-ATPase inhibitors that uncouple the proton transport and ATPase activity of the pump. Conclusion: Residues at the tether connecting V 0 subunit a to the membrane give uncoupling potential to V-ATPases. Significance: The tether may offer new mechanisms to regulate V-ATPase and cellular pH in vivo by uncoupling the pump.

Background: Here we describe superparamagnetic relaxometry (SPMR), a technology that utilizes highly sensitive magnetic sensors and superparamagnetic nanoparticles for cancer detection. Using SPMR, we sensitively and specifically detect nanoparticles conjugated to biomarkers for various types of cancer. SPMR offers high contrast in vivo, as there is no superparamagnetic background, and bones and tissue are transparent to the magnetic fields. Methods: In SPMR measurements, a brief magnetizing pulse is used to align superparamagnetic nanoparticles of a discrete size. Following the pulse, an array of superconducting quantum interference detectors (SQUID) sensors detect the decaying magnetization field. NP size is chosen so that, when bound, the induced field decays in seconds. They are functionalized with specific biomarkers and incubated with cancer cells in vitro to determine specificity and cell binding. For in vivo experiments, functionalized

Purpose of review This review highlights recent findings about the known DNA repair machinery, its impact on chromosomal translocation mechanisms and their relevance to leukemia in the clinic. Recent findings Chromosomal translocations regulate the behavior of leukemia. They not only predict outcome but they define therapy. There is a great deal of knowledge on the products of leukemic translocations, yet little is known about the mechanism by which those translocations occur. Given the large number of DNA double-strand breaks that occur during normal progression through the cell cycle, especially from V(D)J recombination, stalled replication forks or failed decatenation, it is surprising that leukemogenic translocations do not occur more frequently. Fortunately, hematopoietic cells have sophisticated repair mechanisms to suppress such translocations. When these defenses fail leukemia becomes far more common, as seen in inherited deficiencies of DNA repair. Analyzing translocation sequences in cellular and animal models, and in human leukemias, has yielded new insights into the mechanisms of leukemogenic translocations. Summary New data from animal models suggest a two hit origin of leukemic translocations, where there must be both a defect in DNA double-strand break repair and a subsequent failure of cell cycle arrest for leukemogenesis.