MDC1 and RNF8 function in a pathway that directs BRCA1-dependent localization of PALB2 required for homologous recombination

Abstract

SummaryThe PALB2 protein is associated with breast cancer susceptibility and Fanconi anemia. Notably, PALB2 is also required for DNA repair by homologous recombination (HR). However, the mechanisms that regulate PALB2, and the functional significance of its interaction with the BRCA1 breast cancer susceptibility protein, are poorly understood. Here, to better understand these processes, we fused PALB2, or the PALB2(L21P) mutant which cannot bind to BRCA1, with the BRCT repeats that are present in, and which localize, BRCA1. Our results yield important insights into the regulation of PALB2 function. Both fusion proteins can bypass BRCA1 to localize to sites of DNA damage. Further, the localized fusion proteins are functional, as determined by their ability to support the assembly of RAD51 foci, even in the absence of the capacity of PALB2 to bind BRCA1. Strikingly, the localized fusion proteins mediate DNA double-strand break (DSB)-initiated HR and resistance to mitomycin C in PALB2-deficient cells. Additionally, we show that the BRCA1-PALB2 heterodimer, rather than the PALB2-PALB2 homodimer, mediates these responses. Importantly, we offer the first insight into how BRCA1-dependent recruitment of PALB2 is integrated with other DNA damage signaling pathways. We find that PALB2 localization depends on the presence of MDC1, RNF8, RAP80 and Abraxas upstream of BRCA1. Thus, PALB2 may link HR to a key ubiquitin-related signaling pathway that responds to DSBs.

PALB2: The hub of a network of tumor suppressors involved in DNA damage responses

“…Subsequently, it was demonstrated that defective localization of a mutant of the PALB2 coiled-coil domain, and of RAD51 downstream, can be rescued by fusion to the BRCT repeats that normally localize BRCA1 [62]. This lends strong support to the conclusion that BRCA1 localizes PALB2.…”

“…Given that PALB2 is recruited in a BRCA1-dependent manner, the question arose as to whether ubiquitin-based signaling recruits PALB2 [62]. This possibility is supported by the finding that PALB2 colocalizes with ubiquitin, detected using the FK2 antibody, following exposure to IR [62]. Similar to the result for BRCA1 [83–85], RNAi-mediated depletion of RNF8 dramatically reduces IR-induced PALB2 foci [62].…”

Male Fertility Defect Associated with Disrupted BRCA1-PALB2 Interaction in Mice

“…1D). Since monomeric PALB2 has been shown to possess higher activity in DNA binding and RAD51-mediated strand invasion in vitro (37) and a monomeric PALB2 recruited to DNA sites by a fused BRCT domain is functional in HR in the cell (38), it appears that loss of BRCA1 binding is likely the cause of the reduced HR activities of the mutant proteins.…”

RNF8 regulates active epigenetic modifications and escape gene activation from inactive sex chromosomes in post-meiotic spermatids

“…5A,B). RAP80, a DDR factor that binds to ubiquitin generated in a RNF8-dependent manner (Mailand et al 2007;Wu et al 2009;Zhang et al 2012) and transmits DDR signaling in somatic cells, is absent on meiotic sex chromosomes; however, it accumulates on both ends of all chromosomes regardless of the genotype of RNF8 (Fig. 5C,D).…”

“…Briefly, cells were grown overnight on glass coverslips prior to transfection with siRNAs directed against LacZ (control) or RNF8 (59-GGACAAUUAUGGACAACAA-39) (Zhang et al 2012). At 48 h after transfection, cells were incubated with 10 mM BrdU for 24 h and then microirradiated (355 nm) in stripes using an ArcturusXT laser capture microdissection system (Invitrogen).…”