Biology subjects

Takahashi, T. S.

Publications and source records attributed to Takahashi, T. S..

2 recordsLinked to original sources

bioRxiv2023

The Atad5 RFC-like complex is the major unloader of proliferating cell nuclear antigen in Xenopus egg extracts

Proliferating cell nuclear antigen (PCNA) is a homo-trimeric clamp complex that serves as the molecular hub for various DNA transactions, including DNA synthesis and post-replicative mismatch repair. Its timely loading and unloading are critical for genome stability. PCNA loading is catalyzed by Replication factor C (RFC) and the Ctf18 RFC-like complex (Ctf18-RLC), and its unloading is catalyzed by Atad5/Elg1-RLC. However, RFC, Ctf18-RLC, and even some subcomplexes of their shared subunits are capable of unloading PCNA in vitro, leaving an ambiguity in the division of labor in eukaryotic clamp dynamics. By using a system that specifically detects PCNA unloading, we show here that Atad5-RLC, which accounts for only approximately 3% of RFC/RLCs, nevertheless provides the major PCNA unloading activity in Xenopus egg extracts. RFC and Ctf18-RLC each account for approximately 40% of RFC/RLCs, while immunodepletion of neither Rfc1 nor Ctf18 detectably affects the rate of PCNA unloading in our system. PCNA unloading is dependent on the ATP-binding motif of Atad5, independent of nicks on DNA and chromatin assembly, and inhibited effectively by PCNA-interacting peptides. These results support a model in which Atad5-RLC preferentially unloads DNA-bound PCNA molecules that are free from their interactors.

molecular biology↗

bioRxiv2023

Unique and shared functions of the Rad9-Hus1-Rad1 and Mre11-Rad50-Nbs1 complexes in ATR checkpoint activation and long-range DNA end resection in Xenopus egg extracts

Sensing and processing of DNA double-strand breaks (DSBs) are vital to genome stability. DSBs are primarily detected by the ATM checkpoint pathway, where the Mre11-Rad50-Nbs1 (MRN) complex serves as the DSB sensor. Subsequent DSB end resection promotes the transition from the ATM to the ATR checkpoint pathway, where replication protein A, MRN, and the Rad9-Hus1-Rad1 (9-1-1) checkpoint clamp serve as the DNA structure sensors. 9-1-1 and MRN recruit Topbp1, a critical checkpoint mediator that activates the ATR kinase. However, how multiple sensors contribute to regulating end resection and checkpoint activation remains ambiguous. Using DNA substrates that mimic extensively resected DSBs, we show here that MRN and 9-1-1 redundantly stimulate Dna2-dependent long-range end resection and ATR activation in Xenopus egg extracts. MRN serves as the loading platform for Dna2, ATM, and Topbp1. In contrast, 9-1-1 is dispensable for bulk Dna2 loading, and Topbp1 loading is interdependent with 9-1-1 in this pathway. ATR facilitates Mre11 phosphorylation and ATM dissociation. Our results delineate the molecular mechanism of and interplay between two redundant pathways that stimulate ATR checkpoint activation and long-range DSB end resection in vertebrates.

biochemistry↗