Identification of a Substrate Recognition Domain in the Replication Stress Response Protein Zinc Finger Ran-binding Domain-containing Protein 3 (ZRANB3)

Abstract

DNA damage and other forms of replication stress can cause replication forks to stall. Replication stress response proteins stabilize and resolve stalled forks by mechanisms that include fork remodeling to facilitate repair or bypass of damaged templates. Several enzymes including SMARCAL1, HLTF, and ZRANB3 catalyze these reactions. SMARCAL1 and HLTF utilize structurally distinct accessory domains attached to an ATPase motor domain to facilitate DNA binding and catalysis of fork remodeling reactions. Here we describe a substrate recognition domain within ZRANB3 that is needed for it to recognize forked DNA structures, hydrolyze ATP, catalyze fork remodeling, and act as a structure-specific endonuclease. Thus, substrate recognition domains are a common feature of fork remodeling, SNF2-family, DNA-dependent ATPases, and our study provides further mechanistic understanding of how these enzymes maintain genome integrity during DNA replication.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 (1). 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 (2, 3). Fork regression may facilitate DNA repair or template switching to bypass the obstruction (3).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), 3 HLTF (helicase-like transcription factor) and ZRANB3 (zinc finger Ran-binding domain containing 3) are replication stress response proteins that catalyze fork remodeling including fork regression (4 -6). The replication stress response is essential to complete replication accurately. Therefore, defects in this response cause human disease (1). For example, bi-allelic loss of function mutations in SMARCAL1 cause Schimke-Immunoosseous Dysplasia (SIOD) (7). SIOD is a developmental disorder characterized by growth defects, immune-deficiency, and renal failure. Recent studies also suggest that SIOD may be a cancer predisposition syndrome (8 -10). HLTF is silenced in colorectal cancer and ZRANB3 is mutated in endometrial cancers suggesting that both may be tumor suppressors (11,12). SMARCAL1 localizes to stalled replication forks through an interaction with RPA (13-16). The RPA interaction also regulates SMARCAL1 enzymatic activity to ensure that it regresses only stalled forks (17, 18). Although HLTF is present at replication forks, it is unclear if it is recruited through a protein-protein interaction or simply by its structure-specific DNA binding activity (19). ZRANB3 is recruited by binding to poly-ubiquitinated PCNA (5).The enzymatic activities of S...

Functions of SMARCAL1, ZRANB3, and HLTF in maintaining genome stability

“…In contrast to SMARCAL1 and other SNF2 family members, ZRANB3 possesses endonuclease activity in addition to its other enzymatic functions (Weston et al , 2012, Badu-Nkansah et al , 2016, Sebesta et al , 2017). The endonuclease activity depends on ATP hydrolysis by the intact motor domain as well as a C-terminal nuclease domain.…”

“…Given its similar biochemical activities to SMARCAL1, the expectation is that ZRANB3 will bind DNA using similar domains. Thus, two groups have looked for a SRD that would confer DNA binding specificity (Yuan et al , 2012, Badu-Nkansah et al , 2016). Initially, a region between amino acids 712-818 was suggested to be a HARP-like SRD based on limited sequence similarity and functional analyses (Yuan et al , 2012).…”

“…Using sequence conservation and secondary structure predictions as guides, we identified a human ZRANB3 SRD consisting of amino acids 721-869 (Badu-Nkansah et al , 2016). As predicted from the SMARCAL1 studies, this SRD is essential for DNA binding, ATPase function, and annealing helicase activity.…”

“…As predicted from the SMARCAL1 studies, this SRD is essential for DNA binding, ATPase function, and annealing helicase activity. Deletions or amino acid substitution mutations in the SRD are sufficient to abolish all ZRANB3 enzymatic activity including its nuclease activity (Badu-Nkansah et al , 2016). Furthermore, the ZRANB3 SRD by itself is able to bind splayed arm DNA but not double or single-stranded DNA indicating that it provides specificity to fork junctions.…”

SMARCAL1 and telomeres: Replicating the troublesome ends

“…Additionally, each of these proteins contains a distinctive substrate recognition domain that likely dictates their different substrate preferences. 4,23,24 Thus, one hypothesis for the differences between these enzymes is that each may be specialized to resolve different types of replication stress. While experimental challenges with different DNA damaging or replication stress-inducing agents can provide some insights, identifying the endogenous sources of replication stress that these enzymes function to resolve is essential.…”