Exclusion from DNA double-strand breaks is a pervasive feature of the hnRNP family

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are abundant RNA-binding proteins that have been linked individually to the DNA double-strand break (DSB) response, but their behaviour at damaged chromatin has only been examined one protein at a time and under disparate conditions. Here, we compare the core hnRNP family side by side by laser microirradiation and live-cell imaging. We find that 13 of 14 members are excluded from damaged chromatin, 10 directly and 3 after a transient phase of recruitment, establishing exclusion rather than accumulation as the predominant behaviour of this family. We characterised this behaviour in detail for hnRNPH1, which has not previously been linked directly to the DSB response. Its exclusion is sustained for several hours and also occurs at nuclease-induced breaks at a defined genomic locus. Importantly, exclusion is independent of the signalling pathways that drive protein recruitment to breaks and of transcriptional elongation, and instead requires the GYR low-complexity domain, with RNA binding contributing redundantly through three quasi-RNA recognition motifs. Finally, tethering hnRNPH1 to breaks through the RNF8 FHA domain selectively impairs 53BP1 accumulation without affecting γ-H2AX, MDC1 or RPA, and hnRNPH1 is excluded from 53BP1 nuclear bodies in unchallenged cells. Together, these findings establish protein exclusion as an active component of the DSB response and show that the presence of an hnRNP at damaged chromatin can determine which repair factors assemble at DNA break sites.