Genome-wide mapping of long-range contacts unveils clustering of DNA double-strand breaks at damaged active genes.

The ability of DNA double-strand breaks (DSBs) to cluster in mammalian cells has been a subject of intense debate in recent years. Here we used a high-throughput chromosome conformation capture assay (capture Hi-C) to investigate clustering of DSBs induced at defined loci in the human genome. The results unambiguously demonstrated that DSBs cluster, but only when they are induced within transcriptionally active genes.

Non-redundant Functions of ATM and DNA-PKcs in Response to DNA Double-Strand Breaks.

DNA double-strand breaks (DSBs) elicit the so-called DNA damage response (DDR), largely relying on ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PKcs), two members of the PI3K-like kinase family, whose respective functions during the sequential steps of the DDR remains controversial. Using the DIvA system (DSB inducible via AsiSI) combined with high-resolution mapping and advanced microscopy, we uncovered that both ATM and DNA-PKcs spread in cis on a confined region surrounding DSBs, independently of the pathway used for repair. However, once recruited, these kinases exhibit non-overlapping functions on end joining and γH2AX domain establishment.

Proton-induced direct and indirect damage of plasmid DNA.

Clustered DNA damage induced by 10, 20 and 30 MeV protons in pBR322 plasmid DNA was investigated. Besides determination of strand breaks, additional lesions were detected using base excision repair enzymes. The plasmid was irradiated in dry form, where indirect radiation effects were almost fully suppressed, and in water solution containing only minimal residual radical scavenger.

Transcriptionally active chromatin recruits homologous recombination at DNA double-strand breaks.

Although both homologous recombination (HR) and nonhomologous end joining can repair DNA double-strand breaks (DSBs), the mechanisms by which one of these pathways is chosen over the other remain unclear. Here we show that transcriptionally active chromatin is preferentially repaired by HR. Using chromatin immunoprecipitation-sequencing (ChIP-seq) to analyze repair of multiple DSBs induced throughout the human genome, we identify an HR-prone subset of DSBs that recruit the HR protein RAD51, undergo resection and rely on RAD51 for efficient repair.

Cohesin protects genes against γH2AX Induced by DNA double-strand breaks.

Chromatin undergoes major remodeling around DNA double-strand breaks (DSB) to promote repair and DNA damage response (DDR) activation. We recently reported a high-resolution map of γH2AX around multiple breaks on the human genome, using a new cell-based DSB inducible system. In an attempt to further characterize the chromatin landscape induced around DSBs, we now report the profile of SMC3, a subunit of the cohesin complex, previously characterized as required for repair by homologous recombination.

Identification of N-terminally truncated stable nuclear isoforms of CDC25B that are specifically involved in G2/M checkpoint recovery.

CDC25B phosphatases must activate cyclin B-CDK1 complexes to restart the cell cycle after an arrest in G2 phase caused by DNA damage. However, little is known about the precise mechanisms involved in this process, which may exert considerable impact on cancer susceptibility and therapeutic responses. Here we report the discovery of novel N-terminally truncated CDC25B isoforms, referred to as ΔN-CDC25B, with an exclusively nuclear and nonredundant function in cell cycle re-initiation after DNA damage.

A screen for deubiquitinating enzymes involved in the G₂/M checkpoint identifies USP50 as a regulator of HSP90-dependent Wee1 stability.

Tight regulation of cell cycle progression is essential for the maintenance of genomic integrity in response to DNA injury. The aim of this study was to identify new deubiquitinating enzymes (DUBs) involved in the regulation of the G₂/M checkpoint. By using an siRNA-based screen to identify DUBs with an inherent ability to enhance a CDC25B-dependent G₂/M checkpoint bypass, we have identified 11 candidates whose invalidation compromises checkpoint stringency.

Unscheduled expression of CDC25B in S-phase leads to replicative stress and DNA damage.

Background: CDC25B phosphatase is a cell cycle regulator that plays a critical role in checkpoint control. Up-regulation of CDC25B expression has been documented in a variety of human cancers, however, the relationships with the alteration of the molecular mechanisms that lead to oncogenesis still remain unclear. To address this issue we have investigated, in model cell lines, the consequences of unscheduled and elevated CDC25B levels.

Moderate variations in CDC25B protein levels modulate the response to DNA damaging agents.

CDC25B, one of the three members of the CDC25 dual-specificity phosphatase family, plays a critical role in the control of the cell cycle and in the checkpoint response to DNA damage. CDC25B is responsible for the initial dephosphorylation and activation of the cyclin-dependent kinases, thus initiating the train of events leading to entry into mitosis. The critical role played by CDC25B is illustrated by the fact that it is specifically required for checkpoint recovery and that unscheduled accumulation of CDC25B is responsible for illegitimate entry into mitosis.

Genotoxic-activated G2-M checkpoint exit is dependent on CDC25B phosphatase expression.

Cell cycle arrest at the G2-M checkpoint is an essential feature of the mechanisms that preserve genomic integrity. CDC25 phosphatases control cell cycle progression by dephosphorylating and activating cyclin-dependent kinase/cyclin complexes. Their activities are, therefore, tightly regulated to modulate cell cycle arrest in response to DNA damage exposure.

Ability of human CDC25B phosphatase splice variants to replace the function of the fission yeast Cdc25 cell cycle regulator.

CDC25 phosphatases are essential and evolutionary-conserved actors of the eukaryotic cell cycle control. To examine and compare the properties of three splicing variants of human CDC25B, recombinant fission yeast strains expressing the human proteins in place of the endogenous Cdc25 were generated and characterized. We report, that the three CDC25B variants: (i) efficiently replace the yeast counterpart in vegetative growth, (ii) partly restore the gamma and UV radiation DNA damage-activated checkpoint, (iii) fail to restore the DNA replication checkpoint activated by hydroxyurea.

Evolutionary conservation of a novel splice variant of the Cds1/CHK2 checkpoint kinase restricted to its regulatory domain.

The Cds1/CHK2 kinase plays a key role in the activation of the G(2) checkpoint after DNA damage. Here we report the existence in fission yeast of a short variant (Sv) of Cds1 that is produced through an alternative splicing mechanism leading to a frame shift and premature termination. This SvCds1 protein consists solely of the regulatory region and lacks the catalytic domain.

A fission yeast strain expressing human CDC25A phosphatase: a tool for selectivity studies of pharmacological inhibitors of CDC25.

Fission yeast is a simple eukaryotic model organism in which many aspects of cell cycle control can be explored. We examined by homologous recombination whether the human CDC25A phosphatase could substitute for the function of the fission yeast Cdc25. We first show: (a).

Human CDC25B and CDC25C differ by their ability to restore a functional checkpoint response after gene replacement in fission yeast.

In fission yeast, inactivation of the Cdc25 phosphatase by checkpoint kinases participates in the signaling cascade that temporarily stops cell cycle progression after DNA damage. In human, CDC25B and C are also known to be targeted by a similar checkpoint machinery. We have examined by homologous recombination, whether CDC25B and CDC25C were able to substitute for the function of fission yeast Cdc25.