Abstract
DNA double-strand breaks (DSB) elicit a ubiquitylation cascade that controls DNA repair pathway choice. This cascade involves the ubiquitylation of histone H2A by the RNF168 ligase and the subsequent recruitment of RIF1, which suppresses homologous recombination (HR) in G1 cells. The RIF1-dependent suppression is relieved in S/G2 cells, allowing PALB2-driven HR to occur. With the inhibitory impact of RIF1 relieved, it remains unclear how RNF168-induced ubiquitylation influences HR. Here, we uncover that RNF168 links the HR machinery to H2A ubiquitylation in S/G2 cells. We show that PALB2 indirectly recognizes histone ubiquitylation by physically associating with ubiquitin-bound RNF168. This direct interaction is mediated by the newly identified PALB2-interacting domain (PID) in RNF168 and the WD40 domain in PALB2, and drives DNA repair by facilitating the assembly of PALB2-containing HR complexes at DSBs. Our findings demonstrate that RNF168 couples PALB2-dependent HR to H2A ubiquitylation to promote DNA repair and preserve genome integrity.
Highlights
Chromosomal DNA double-strand breaks (DSB) that arise in S phase during DNA replication or early in G2 phase are removed by the homologous recombination (HR) machinery, which utilizes the intact genetic information from the sister chromatid as a template for repair
While BRCA1 depletion completely abolished PALB2 recruitment (Zhang et al, 2009a, 2009b), loss of RAP80 had no appreciable impact (Figure 2A,D and Figure 2—figure supplement 1A,B). These results suggest that RNF8/RNF168 contribute to the BRCA1-dependent loading of PALB2 at DSBs independently of RAP80, which is in accordance with our tethering results (Figure 1F,G), and consistent with several studies showing that RAP80 does not contribute to, or may even antagonize HR (Coleman and Greenberg, 2011; Hu et al, 2011; Typas et al, 2015)
We demonstrate that RNF168 drives the recruitment of PALB2 to DSBs in a manner depending on its ubiquitin ligase activity toward histone H2A on K13/K15
Summary
Chromosomal DNA double-strand breaks (DSB) that arise in S phase during DNA replication or early in G2 phase are removed by the homologous recombination (HR) machinery, which utilizes the intact genetic information from the sister chromatid as a template for repair. Following their detection, the ends of a DSB are resected to generate extended 3’ single-stranded DNA (ssDNA) overhangs, which are bound by the ssDNA-binding protein RPA. PALB2 forms oligomers to interact with the breast cancer susceptibility proteins 1 and 2 (BRCA1 and BRCA2) and the recombinase RAD51 (Sy et al, 2009; Zhang et al, 2009a, 2009b; Buisson and Masson, 2012). Once recruited to resected DSB ends by BRCA1, PALB2 facilitates the assembly of BRCA2 and RAD51 onto broken DNA ends, the latter of which catalyzes strand
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