Untangling the crosstalk between BRCA1 and R-loops during DNA repair

R-loops are RNA:DNA hybrids assembled during biological processes but are also linked to genetic instability when formed out of their natural context. Emerging evidence suggests that the repair of DNA double-strand breaks requires the formation of a transient R-loop, which eventually must be removed to guarantee a correct repair process. The multifaceted BRCA1 protein has been shown to be recruited at this specific break-induced R-loop, and it facilitates mechanisms in order to regulate R-loop removal. In this review, we discuss the different potential roles of BRCA1 in R-loop homeostasis during DNA repair and how these processes ensure faithful DSB repair.Genome Instability and Cance

Two redundant ubiquitin-dependent pathways of BRCA1 localization to DNA damage sites

The tumor suppressor BRCA1 accumulates at sites of DNA damage in a ubiquitin-dependent manner. In this work, we revisit the role of RAP80 in promoting BRCA1 recruitment to damaged chromatin. We find that RAP80 acts redundantly with the BRCA1 RING domain to promote BRCA1 recruitment to DNA damage sites. We show that that RNF8 E3 ligase acts upstream of both the RAP80- and RING-dependent activities, whereas RNF168 acts uniquely upstream of the RING domain. BRCA1 RING mutations that do not impact BARD1 interaction, such as the E2 binding-deficient I26A mutation, render BRCA1 unable to accumulate at DNA damage sites in the absence of RAP80. Cells that combine BRCA1 I26A and mutations that disable the RAP80-BRCA1 interaction are hypersensitive to PARP inhibition and are unable to form RAD51 foci. Our results suggest that in the absence of RAP80, the BRCA1 E3 ligase activity is necessary for recognition of histone H2A Lys13/Lys15 ubiquitylation by BARD1, although we cannot rule out the possibility that the BRCA1 RING facilitates ubiquitylated nucleosome recognition in other ways.Genome Instability and Cance

ELOF1 is a transcription-coupled DNA repair factor that directs RNA polymerase II ubiquitylation

Two side-by-side papers report that the transcription elongation factor ELOF1 drives transcription-coupled repair and prevents replication stress.Cells employ transcription-coupled repair (TCR) to eliminate transcription-blocking DNA lesions. DNA damage-induced binding of the TCR-specific repair factor CSB to RNA polymerase II (RNAPII) triggers RNAPII ubiquitylation of a single lysine (K1268) by the CRL4(CSA) ubiquitin ligase. How CRL4(CSA) is specifically directed towards K1268 is unknown. Here, we identify ELOF1 as the missing link that facilitates RNAPII ubiquitylation, a key signal for the assembly of downstream repair factors. This function requires its constitutive interaction with RNAPII close to K1268, revealing ELOF1 as a specificity factor that binds and positions CRL4(CSA) for optimal RNAPII ubiquitylation. Drug-genetic interaction screening also revealed a CSB-independent pathway in which ELOF1 prevents R-loops in active genes and protects cells against DNA replication stress. Our study offers key insights into the molecular mechanisms of TCR and provides a genetic framework of the interplay between transcriptional stress responses and DNA replication.Cancer Signaling networks and Molecular Therapeutic

Orchestrating the Ubiquitin solos in the hematopoietic symphony. Genomic & proteomic studies on ubiquitination in normal and malignant blood cell development

Contains fulltext : 93616.pdf (publisher's version ) (Open Access)Radboud Universiteit Nijmegen, 22 juni 2012Promotor : Witte, T.J.M. de Co-promotores : Reijden, B.A. van der, Jansen, J.H.179 p

High DNA-methyltransferase 3B expression predicts poor outcome in acute myeloid leukemia, especially among patients with co-occurring NPM1 and FLT3 mutations

Contains fulltext : 138167.pdf (publisher's version ) (Open Access

PARP Inhibitor Resistance: A Tug-of-War in BRCA-Mutated Cells

Poly-(ADP)-ribose polymerase (PARP) inhibition is synthetic lethal with deficiency for homologous recombination (HR), a pathway essential for DNA double-strand break repair. PARP inhibitors (PARPi) therefore hold great promise for the treatment of tumors with disruptive mutations in BRCA1/2 or other HR factors. Unfortunately, PARPi resistance has proved to be a major problem in the clinic. Knowledge about PARPi resistance is expanding quickly, revealing four main mechanisms that alter drug availability, affect (de)PARylation enzymes, restore HR, or restore replication fork stability. We discuss how studies on resistance mechanisms have yielded important insights into the regulation of DNA double-strand break (DSB) repair and replication fork protection, and how these studies could pave the way for novel treatment options to target resistance mechanisms or acquired vulnerabilities.Genome Instability and Cance