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DNA double-strand breaks (DSBs) are highly toxic lesions that threaten genome integrity and cell survival. To avoid harmful repercussions of DSBs, a wide variety of DNA repair factors are recruited to execute DSB repair. Previously, we demonstrated that RBM6 splicing factor facilitates homologous recombination (HR) of DSB by regulating alternative splicing-coupled nonstop-decay of the HR protein APBB1/Fe65. Here, we describe a splicing-independent function of RBM6 in promoting HR repair of DSBs. We show that RBM6 is recruited to DSB sites and PARP1 activity indirectly regulates RBM6 recruitment to DNA breakage sites. Deletion mapping analysis revealed a region containing five glycine residues within the G-patch domain that regulates RBM6 accumulation at DNA damage sites. We further ascertain that RBM6 interacts with Rad51, and this interaction is attenuated in RBM6 mutant lacking the G-patch domain (RBM6del(G-patch)). Consequently, RBM6del(G-patch) cells exhibit reduced levels of Rad51 foci after ionizing radiation. In addition, while RBM6 deletion mutant lacking the G-patch domain has no detectable effect on the expression levels of its splicing targets Fe65 and Eya2, it fails to restore the integrity of HR. Altogether, our results suggest that RBM6 recruitment to DSB promotes HR repair, irrespective of its splicing activity. PARP1 activity indirectly regulates RBM6 recruitment to DNA damage sites. Five glycine residues within the G-patch domain of RBM6 are critical for its recruitment to DNA damage sites, but dispensable for its splicing activity. RBM6 G-patch domain fosters its interaction with Rad51 and promotes Rad51 foci formation following irradiation. RBM6 recruitment to DSB sites underpins HR repair. [ABSTRACT FROM AUTHOR]

Previously, we showed that CDYL1 is recruited to DNA double-strand breaks (DSBs) to promote homologous recombination (HR) repair and foster transcriptional silencing. However, how CDYL1 elicits DSB-induced silencing is not fully understood. Here, we identify a CDYL1-dependent local decrease in the transcriptionally active marks histone lysine crotonylation (Kcr) and crotonylated lysine 9 of H3 (H3K9cr) at Asi SI-induced DSBs, which correlates with transcriptional silencing. Mechanistically, we reveal that CDYL1 crotonyl-CoA hydratase activity counteracts Kcr and H3K9cr at DSB sites, which triggers the eviction of the transcription elongation factor ENL and fosters transcriptional silencing. Furthermore, genetic inhibition of CDYL1 hydratase activity blocks the reduction in H3K9cr and alleviates DSB-induced silencing, whereas HR efficiency unexpectedly remains intact. Therefore, our results functionally uncouple the repair and silencing activity of CDYL1 at DSBs. In a broader context, we address a long-standing question concerning the functional relationship between HR repair and DSB-induced silencing, suggesting that they may occur independently. [Display omitted] • Local decrease in Kcr at DSBs correlates with transcriptional silencing • CDYL1 crotonyl-CoA hydratase activity downregulates Kcr at DSBs • Kcr reduction at DSBs promotes ENL eviction and fosters transcriptional silencing • CDYL1 roles in DSB-induced silencing and HR repair are functionally uncoupled Abu-Zhaiya et al. identify a CDYL1-dependent decrease in lysine crotonylation at DSB sites that fosters transcriptional silencing. It is widely accepted that DSB-induced silencing is prerequisite for timely DSB repair. Here, the authors challenge the current dogma and provide evidence that functionally uncouple DSB-induced silencing and repair, suggesting that they occur independently. [ABSTRACT FROM AUTHOR]