LC3B drives transcription-associated homologous recombination via direct interaction with R-loops.

Exploring the connection between ubiquitin-like modifiers (ULMs) and the DNA damage response (DDR), we employed several advanced DNA damage and repair assay techniques and identified a crucial role for LC3B. Notably, its RNA recognition motif (RRM) plays a pivotal role in the context of transcription-associated homologous recombination (HR) repair (TA-HRR), a particular subset of HRR pathways. Surprisingly, independent of autophagy flux, LC3B interacts directly with R-loops at DNA lesions within transcriptionally active sites via its RRM, promoting TA-HRR. Using native RNA immunoprecipitation (nRIP) coupled with high-throughput sequencing (nRIP-seq), we discovered that LC3B also directly interacts with the 3'UTR AU-rich elements (AREs) of BRCA1 via its RRM, influencing its stability. This suggests that LC3B regulates TA-HRR both proximal to and distal from DNA lesions. Data from our LC3B depletion experiments showed that LC3B knockdown disrupts end-resection for TA-HRR, redirecting it towards the non-homologous end joining (NHEJ) pathway and leading to chromosomal instability, as evidenced by alterations in sister chromatid exchange (SCE) and interchromosomal fusion (ICF). Thus, our findings unveil autophagy-independent functions of LC3B in DNA damage and repair pathways, highlighting its importance. This could reshape our understanding of TA-HRR and the interaction between autophagy and DDR.

USP39 promotes non-homologous end-joining repair by poly(ADP-ribose)-induced liquid demixing.

Mutual crosstalk among poly(ADP-ribose) (PAR), activated PAR polymerase 1 (PARP1) metabolites, and DNA repair machinery has emerged as a key regulatory mechanism of the DNA damage response (DDR). However, there is no conclusive evidence of how PAR precisely controls DDR. Herein, six deubiquitinating enzymes (DUBs) associated with PAR-coupled DDR were identified, and the role of USP39, an inactive DUB involved in spliceosome assembly, was characterized. USP39 rapidly localizes to DNA lesions in a PAR-dependent manner, where it regulates non-homologous end-joining (NHEJ) via a tripartite RG motif located in the N-terminus comprising 46 amino acids (N46). Furthermore, USP39 acts as a molecular trigger for liquid demixing in a PAR-coupled N46-dependent manner, thereby directly interacting with the XRCC4/LIG4 complex during NHEJ. In parallel, the USP39-associated spliceosome complex controls homologous recombination repair in a PAR-independent manner. These findings provide mechanistic insights into how PAR chains precisely control DNA repair processes in the DDR.