SUMMARY Defective DNA repair by homologous recombination (HR) is thought to be a major contributor to tumorigenesis in individuals carrying Brca1 mutations. Here we show that DNA breaks in Brca1-deficient cells are aberrantly joined into complex chromosome rearrangements by a process dependent on the non-homologous end joining (NHEJ) factors, 53BP1 and DNA Ligase 4. Loss of 53BP1 alleviates hypersensitivity of Brca1 mutant cells to PARP inhibition and restores error-free repair by HR. Mechanistically, 53BP1 deletion promotes ATM-dependent processing of broken DNA ends to produce recombinogenic single-stranded DNA competent for HR. In contrast, Lig4 deficiency does not rescue the HR defect in Brca1 mutant cells, but prevents the joining of chromatid breaks into chromosome rearrangements. Our results illustrate that HR and NHEJ compete to process DNA breaks that arise during DNA replication, and that shifting the balance between these pathways can be exploited to selectively protect or kill cells harboring Brca1 mutations.

The progressive accumulation of DNA damage is thought to be one of the driving forces that initiates ageing. However, the nature of the damage that arises endogenously is still ill-defined. A known source of endogenous damage is replicative stress (RS), which is intrinsically associated to DNA replication and prevented mainly by the ATR kinase. Here, we have developed a murine model of the human Seckel Syndrome characterized by a severe deficiency in ATR. Seckel mice suffer high levels of RS during embryogenesis when proliferation is widespread, but which decrease to marginal levels in postnatal life. In spite of this decrease, adult Seckel mice present accelerated ageing, which is further aggravated in the absence of p53 due to a further increase of RS. Together, these results support the concept that endogenous RS, particularly in utero, contributes to the onset of ageing in postnatal life and this is counterbalanced by the RS-limiting role of the checkpoint proteins ATR and p53.

Summary 53BP1 is a DNA damage protein that forms phosphorylated H2AX (γ-H2AX) dependent foci in a 1 Mb region surrounding DNA double strand breaks (DSBs). In addition, 53BP1 promotes genomic stability by regulating the metabolism of DNA ends. We have compared the joining rates of paired DSBs separated by 1.2 kb to 27 Mb on chromosome 12 in the presence or absence of 53BP1. 53BP1 facilitates joining of intrachromosomal DSBs but only at distances corresponding to γ-H2AX spreading. In contrast, DNA end protection by 53BP1 is distance independent. Furthermore, analysis of 53BP1 mutants shows that chromatin association, oligomerization, and N-terminal ATM phosphorylation are all required for DNA end protection and joining as measured by immunoglobulin class switch recombination. The data elucidate the molecular events that are required for 53BP1 to maintain genomic stability and point to a model wherein 53BP1 and H2AX cooperate to repress resection of DSBs.