Mechanisms of DNA Translocases in the Repair of Damaged Replication Forks

Abstract

Genomic replication is a highly challenging task. The DNA replication machinery must precisely duplicate billions of base pairs while tolerating a multitude of obstacles including damaged DNA, collisions with transcriptional machineries, unusual DNA structures and other difficult to replicate sequences. Many of these obstacles stall replication forks and activate replication stress responses that stabilize and restart persistently stalled forks. These mechanisms include fork remodeling to regress replication forks into a chicken foot DNA structure. Fork regression may facilitate DNA repair or template switching to bypass the obstruction. Several members of the SNF2 family of DNA-dependent ATPases including SMARCAL1 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A-like protein 1), HLTF (Helicase like transcription factor) and ZRANB3 (Zinc finger Ran-binding protein 2- type containing 3) are replication stress response proteins that catalyze fork remodeling including fork regression. The enzymatic activities of SMARCAL1 and HLTF are dependent on a SNF2 ATPase motor domain and a substrate recognition domain (SRD) that is thought to mediate binding to specific structures at stalled replication forks. The SRD of SMARCAL1 is its HARP2 domain, which is required for SMARCAL1 binding to branched DNA structures as well as its DNA-dependent ATPase and fork regression activities. The SRD in HLTF is its HIRAN domain, which is unrelated in sequence and structure to the HARP domain and interacts with the exposed 3’ end of small DNA flaps. The HIRAN domain is also important for HLTF mediated fork regression activity. Interestingly, unlike SMARCAL1 and HLTF, ZRANB3 contains a highly conserved ATP-dependent, substrate specific HNH endonuclease domain and catalyzes nuclease activity to branched DNA substrates. How ZRANB3 catalyzes fork remodeling and endonuclease activities is unknown. This work identified a substrate recognition domain within ZRANB3 that is needed for it to recognize and bind forked DNA structures, hydrolyze ATP, and catalyze fork remodeling and endonuclease activities. Importantly, this work provides a mechanistic understanding of how these enzymes operate within the replication stress response to restart stalled replication forks.