During the evolution of cancer, the incipient tumour experiences 'oncogenic stress', which evokes a counter-response to eliminate such hazardous cells. However, the nature of this stress remains elusive, as does the inducible anti-cancer barrier that elicits growth arrest or cell death. Here we show that in clinical specimens from different stages of human tumours of the urinary bladder, breast, lung and colon, the early precursor lesions (but not normal tissues) commonly express markers of an activated DNA damage response. These include phosphorylated kinases ATM and Chk2, and phosphorylated histone H2AX and p53. Similar checkpoint responses were induced in cultured cells upon expression of different oncogenes that deregulate DNA replication. Together with genetic analyses, including a genome-wide assessment of allelic imbalances, our data indicate that early in tumorigenesis (before genomic instability and malignant conversion), human cells activate an ATR/ATM-regulated DNA damage response network that delays or prevents cancer. Mutations compromising this checkpoint, including defects in the ATM-Chk2-p53 pathway, might allow cell proliferation, survival, increased genomic instability and tumour progression.

In the S and G2 phases of the cell cycle, DNA double-strand breaks (DSBs) are processed into single-stranded DNA, triggering ATR-dependent checkpoint signaling and DSB repair by homologous recombination (HR). Previous work has implicated the MRE11 complex in such DSB processing events. Here, we show that the human CtIP protein confers resistance to DSB-inducing agents and is recruited to DSBs exclusively in S/G2. Moreover, we reveal that CtIP is required for DSB resection, and thereby for recruitment of RPA and ATR to DSBs and ensuing ATR activation. Furthermore, we establish that CtIP physically and functionally interacts with the MRE11 complex, and that both CtIP and MRE11 are required for efficient HR. Finally, we reveal that CtIP displays sequence homology with Sae2, which is involved in MRE11-dependent DSB processing in yeast. These findings establish evolutionarily conserved roles for CtIP-like proteins in controlling DSB resection, checkpoint signaling and HR.DSBs are highly cytotoxic lesions induced by ionizing radiation and certain anti-cancer drugs. They also arise when replication forks encounter other lesions and are generated as intermediates during meiotic recombination1. Cells possess two main DSB repair mechanisms: non-homologous end-joining (NHEJ) and homologous recombination (HR). While NHEJ predominates in G0/G1 and is error-prone, HR is restricted to S and G2, when sister chromatids allow faithful repair2-4. HR is initiated by resection of DSBs to generate single-stranded DNA (ssDNA) that binds RPA. A ssDNA-RAD51 nucleoprotein filament then forms to initiate strand invasion. ssDNA-RPA also recruits the protein kinase ATR, triggering ATR-dependent checkpoint signaling by the protein kinase Chk15. As DSB resection is largely restricted to S and G2, both HR and checkpoint signaling are subject to cell-cycle control6-8.A factor implicated in DSB resection is the MRE11-RAD50-NBS1 (MRN) complex, which binds DNA ends, possesses exo-and endo-nuclease activities and functions in triggeringCorrespondence and requests for materials should be addressed to: Stephen P. Jackson 1 , Email: s.jackson@gurdon.cam.ac.uk, Telephone: +44 (0)1223 334088, Fax: +44 (0)1223 334089. Author contributions S.F and R.B generated CtIP cDNA, CtIP antibodies and recombinant CtIP protein. C.L., J.L., M.M and J.B generated the cell lines with GFP-tagged proteins, conceived, performed and evaluated the real-time imaging experiments, and performed the HR measurements. All other experiments were conceived by A.A.S and S.P.J, and were performed by A.A.S with the help of J.C. A.A.S and S.P.J wrote the paper. All authors discussed the results and commented on the manuscript. Author informationThe authors declare no competing financial interest. Europe PMC Funders Group CtIP affects cellular responses to DSBsTo investigate CtIP function, we examined how its depletion affected clonogenic survival of human U2OS cells following their treatment with DNA-damaging agents. To circumvent possible effects arising from CtIP's involveme...

Accumulation of repair proteins on damaged chromosomes is required to restore genomic integrity. However, the mechanisms of protein retention at the most destructive chromosomal lesions, the DNA double-strand breaks (DSBs), are poorly understood. We show that RNF8, a RING-finger ubiquitin ligase, rapidly assembles at DSBs via interaction of its FHA domain with the phosphorylated adaptor protein MDC1. This is accompanied by an increase in DSB-associated ubiquitylations and followed by accumulation of 53BP1 and BRCA1 repair proteins. Knockdown of RNF8 or disruption of its FHA or RING domains impaired DSB-associated ubiquitylation and inhibited retention of 53BP1 and BRCA1 at the DSB sites. In addition, we show that RNF8 can ubiquitylate histone H2A and H2AX, and that its depletion sensitizes cells to ionizing radiation. These data suggest that MDC1-mediated and RNF8-executed histone ubiquitylation protects genome integrity by licensing the DSB-flanking chromatin to concentrate repair factors near the DNA lesions.

DNA double-strand breaks (DSBs) not only interrupt the genetic information, but also disrupt the chromatin structure, and both impairments require repair mechanisms to ensure genome integrity. We showed previously that RNF8-mediated chromatin ubiquitylation protects genome integrity by promoting the accumulation of repair factors at DSBs. Here, we provide evidence that, while RNF8 is necessary to trigger the DSB-associated ubiquitylations, it is not sufficient to sustain conjugated ubiquitin in this compartment. We identified RNF168 as a novel chromatin-associated ubiquitin ligase with an ability to bind ubiquitin. We show that RNF168 interacts with ubiquitylated H2A, assembles at DSBs in an RNF8-dependent manner, and, by targeting H2A and H2AX, amplifies local concentration of lysine 63-linked ubiquitin conjugates to the threshold required for retention of 53BP1 and BRCA1. Thus, RNF168 defines a new pathway involving sequential ubiquitylations on damaged chromosomes and uncovers a functional cooperation between E3 ligases in genome maintenance.

It is generally thought that the DNA-damage checkpoint kinases, ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR), work independently of one another. Here, we show that ATM and the nuclease activity of meiotic recombination 11 (Mre11) are required for the processing of DNA double-strand breaks (DSBs) to generate the replication protein A (RPA)-coated ssDNA that is needed for ATR recruitment and the subsequent phosphorylation and activation of Chk1. Moreover, we show that efficient ATM-dependent ATR activation in response to DSBs is restricted to the S and G2 cell cycle phases and requires CDK kinase activity. Thus, in response to DSBs, ATR activation is regulated by ATM in a cell-cycle dependent manner.