The opposing activities of 53BP1 and BRCA1 influence pathway choice of DNA double-strand break repair. How BRCA1 counters the inhibitory effect of 53BP1 on DNA resection and homologous recombination is unknown. Here we identify the site of BRCA1-BARD1 required for priming ubiquitin transfer from E2~ubiquitin. We demonstrate that BRCA1-BARD1's ubiquitin ligase activity is required for repositioning 53BP1 on damaged chromatin. We confirm H2A ubiquitylation by BRCA1-BARD1 and show that an H2A-ubiquitin fusion protein promotes DNA resection and repair in BARD1 deficient cells. We show BRCA1-BARD1 function in homologous recombination requires the chromatin remodeler SMARCAD1. SMARCAD1 binding to H2A-ubiquitin, optimal localization to sites of damage and activity in DNA repair requires its ubiquitin-binding CUE domains. SMARCAD1 is required for 53BP1 repositioning and the need for SMARCAD1 in Olaparib or camptothecin resistance is alleviated by 53BP1 loss. Thus BRCA1-BARD1 ligase activity and subsequent SMARCAD1-dependent chromatin remodeling are critical regulators of DNA repair.Introduction.

Genomic integrity is constantly threatened by problems encountered by the replication fork. BRCA1, BRCA2 and a subset of Fanconi Anaemia proteins protect stalled replication forks from nuclease degradation through pathways involving RAD51. The contribution and regulation of BRCA1 in replication fork protection, and whether this relates to, or differs from, BRCA1's role in homologous recombination (HR) is not clear. Here we show that the canonical BRCA1-PALB2 interaction is not required for fork protection but instead BRCA1-BARD1 is regulated through a conformational change mediated by the phosphorylation-directed prolyl isomerase, PIN1. PIN1 activity enhances BRCA1-BARD1 interaction with RAD51 and consequently RAD51's presence at stalled replication structures. We identify patient missense variants in the regulated BRCA1-BARD1 regions which show poor nascent strand protection but remain proficient for HR, defining novel domains required for fork protection associated with cancer development. Together these findings reveal a previously unrecognised pathway that governs BRCA1-mediated replication fork protection. Main Text Fork progression can be slowed by conflicts with transcription, deoxyribonucleotide (dNTP) shortage or by difficult to replicate sequences, frequently causing fork stalling 1. In order to prevent stalled forks collapsing into DNA double strand breaks (DSBs), a number of responses are elicited including fork remodelling and subsequent nascent strand protection. Agents that cause replicative stress or compromise DNA Polymerase-α function result in a proportion of forks reversing (reviewed in 2,3). The regressed arm of nascent DNA in reversed forks resembles a single-ended DNA DSB which is protected from excessive resection by RAD51. Several factors contribute to RAD51-mediated fork protection including BRCA1/2, FANCA/D2, RAD51 paralogs, BOD1L, SETD1A, WRNIP and Abro1 2 .

Diacylglycerol kinase (DGK)α converts diacylglycerol to phosphatidic acid. This lipid kinase sustains survival, migration and invasion of tumor cells, with no effect over untransformed cells, suggesting its potential as a cancer-specific target. Nonetheless the mechanisms that underlie DGKα specific contribution to cancer survival have not been elucidated. Using three-dimensional (3D) colon and breast cancer cell cultures, we demonstrate that DGKα upregulation is part of the transcriptional program that results in Src activation in these culture conditions. Pharmacological or genetic DGKα silencing impaired tumor growth in vivo confirming its function in malignant transformation. DGKα-mediated Src regulation contributed to limit the effect of Src inhibitors, and its transcriptional upregulation in response to PI3K/Akt inhibitors resulted in reduced toxicity. Src oncogenic properties and contribution to pharmacological resistance have been linked to its overactivation in cancer. DGKα participation in this central node helps to explain why its pharmacological inhibition or siRNA-mediated targeting specifically alters tumor viability with no effect on untransformed cells. Our results identify DGKα-mediated stabilization of Src activation as an important mechanism in tumor growth, and suggest that targeting this enzyme, alone or in combination with other inhibitors in wide clinical use, could constitute a treatment strategy for aggressive forms of cancer.

Accurate chromosomal DNA replication is essential to maintain genomic stability. Genetic evidence suggests that certain repetitive sequences impair replication, yet the underlying mechanism is poorly defined. Replication could be directly inhibited by the DNA template or indirectly, for example by DNA-bound proteins. Here, we reconstitute replication of mono-, di- and trinucleotide repeats in vitro using eukaryotic replisomes assembled from purified proteins. We find that structure-prone repeats are sufficient to impair replication. Whilst template unwinding is unaffected, leading strand synthesis is inhibited, leading to fork uncoupling. Synthesis through hairpin-forming repeats is rescued by replisome-intrinsic mechanisms, whereas synthesis of quadruplex-forming repeats requires an extrinsic accessory helicase. DNA-induced fork stalling is mechanistically similar to that induced by leading strand DNA lesions, highlighting structure-prone repeats as an important potential source of replication stress. Thus, we propose that our understanding of the cellular response to replication stress may also be applied to DNA-induced replication stalling.