Structural insights into the function of ZRANB3 in replication stress response

Abstract

Strategies to resolve replication blocks are critical for the maintenance of genome stability. Among the factors implicated in the replication stress response is the ATP-dependent endonuclease ZRANB3. Here, we present the structure of the ZRANB3 HNH (His-Asn-His) endonuclease domain and provide a detailed analysis of its activity. We further define PCNA as a key regulator of ZRANB3 function, which recruits ZRANB3 to stalled replication forks and stimulates its endonuclease activity. Finally, we present the co-crystal structures of PCNA with two specific motifs in ZRANB3: the PIP box and the APIM motif. Our data provide important structural insights into the PCNA-APIM interaction, and reveal unexpected similarities between the PIP box and the APIM motif. We propose that PCNA and ATP-dependency serve as a multi-layered regulatory mechanism that modulates ZRANB3 activity at replication forks. Importantly, our findings allow us to interpret the functional significance of cancer associated ZRANB3 mutations.

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.…”

“…Some cancer associated mutations in ZRANB3 inactivate its nuclease activity without affecting its ATPase activity (Sebesta et al , 2017), and the nuclease domain contributes to ZRANB3 localization to damaged forks (Weston et al , 2012). Thus, nuclease activity may be important for its genome protection functions, but further studies will be needed to determine if nuclease inactivation actually drives tumorigenesis.…”

“…Interestingly, ZRANB3 is the only protein present in vertebrates described to contain this domain. Further, structure comparisons of the HNH of ZRANB3 with other HNH domains from lower organisms indicate that ZRANB3 contains an evolutionarily conserved unique sequence insert in the HNH domain that is absent in most other organisms (Sebesta et al , 2017). Deletions in this ZRANB3-specific region abolish nuclease activity.…”

“…This structure could be important for recognition of ZRANB3-specific DNA substrates for cleavage, but future studies are needed to confirm this hypothesis. Importantly, the nuclease activity conferred by the ZRANB3 HNH domain is dependent on an intact motor domain, ATP hydrolysis, and an intact SRD domain (Weston et al , 2012, Badu-Nkansah et al , 2016, Sebesta et al , 2017). How this linkage is achieved is not known.…”

Simultaneous dual-channel imaging to quantify interdependent protein recruitment to laser-induced DNA damage sites

“…As a ring-shaped homotrimer, PCNA encircles and slides along DNA, and recruits proteins via the interdomain connector loop on the outer surface serving as the common PCNA-protein interaction interface [14]. Structure-specific endonuclease ZRANB3, exonuclease EXO1, DNA methyl transferase DNMT1, replication licensing factor CDT1, PCNA loader RFC1 and PARG bind PCNA via the PCNA-interacting protein motif (PIP-box), which also mediates their recruitment to laser-induced DNA damage sites in a PCNA-dependent fashion [35–42]. In the case of EXO1 and PARG, PARP1 promotes early recruitment, while PCNA is responsible for their retention at DNA damage sites [36–38].…”