The SPATA5-SPATA5L1 ATPase complex directs replisome proteostasis to ensure genome integrity

Ubiquitin-dependent unfolding of the CMG helicase by VCP/p97 is required to terminate DNA replication. Other replisome components are not processed in the same fashion, suggesting that additional mechanisms underlie replication protein turnover. Here, we identify replisome factor interactions with a protein complex composed of AAA+ ATPases SPATA5-SPATA5L1 together with heterodimeric partners C1orf109-CINP (55LCC). An integrative structural biology approach revealed a molecular architecture of SPATA5-SPATA5L1 N-terminal domains interacting with C1orf109-CINP to form a funnel-like structure above a cylindrically shaped ATPase motor. Deficiency in the 55LCC complex elicited ubiquitin-independent proteotoxicity, replication stress, and severe chromosome instability. 55LCC showed ATPase activity that was specifically enhanced by replication fork DNA and was coupled to cysteine protease-dependent cleavage of replisome substrates in response to replication fork damage. These findings define 55LCC-mediated proteostasis as critical for replication fork progression and genome stability and provide a rationale for pathogenic variants seen in associated human neurodevelopmental disorders. [Display omitted] •SPATA5 and SPATA5L1 form a heterohexameric complex with C1orf109-CINP (55LCC)•55LCC associates with chromatin and exhibits DNA-stimulated ATPase activity•55LCC loss causes proteotoxicity, replication stress, and genome instability•55LCC processes replisome substrates in response to replication fork insults The 55LCC heterohexameric ATPase complex promotes replisome proteostasis to maintain replication fork progression and genome stability.