Chracterization of Pif1 Helicase

Pif1 is a multifunctional DNA helicase crucial for maintaining both nuclear and mitochondrial (mt) DNA. Although Pif1 is not essential for normal S-phase replication, nuclear Pif1 plays multiple roles. For example, it negatively regulates telomerase at telomeres and double-strand breaks, participates in Okazaki fragment processing, and suppresses genome instability at G-quadruplex motifs. Most importantly, Pif1 is critical for break-induced replication (BIR), driving extensive (long-tract) BIR synthesis. Pif1 is thought to have two functions during BIR: 1) unwinding the parent DNA duplex ahead of the D-loop and 2) dissociating the newly synthesized DNA from its template. Given its essential role in BIR, it is important to identify and characterize the functional domains of Pif1.

Pif1 consists of three domains: the amino-terminal (N-terminus) domain, the central helicase domain, and the carboxy-terminal (C-terminus) domain. While the central helicase domain is well-characterized, both the amino- and carboxy-terminal domains remain poorly understood. Recently, our lab has observed that phosphorylation of Pif1 at the N-terminus, specifically at five serine residues, is crucial for its nuclear activities, including BIR. This project aims to investigate how phosphorylation at the N-terminus regulates Pif1's nuclear functions, particularly during BIR.