Deciphering the mechanism of processive ssDNA digestion by the Dna2-RPA ensemble

Jiangchuan Shen,Jonathan Trinidad,Grzegorz Ira,Nhung Tuyet Pham,Zhi Qi,Yiling Zhao,Yixiang Zhang,Hengyao Niu,Yuxi Li

doi:10.1038/s41467-021-27940-y

Abstract

Single-stranded DNA (ssDNA) commonly occurs as intermediates in DNA metabolic pathways. The ssDNA binding protein, RPA, not only protects the integrity of ssDNA, but also directs the downstream factor that signals or repairs the ssDNA intermediate. However, it remains unclear how these enzymes/factors outcompete RPA to access ssDNA. Using the budding yeast Saccharomyces cerevisiae as a model system, we find that Dna2 — a key nuclease in DNA replication and repair — employs a bimodal interface to act with RPA both in cis and in trans. The cis-activity makes RPA a processive unit for Dna2-catalyzed ssDNA digestion, where RPA delivers its bound ssDNA to Dna2. On the other hand, activity in trans is mediated by an acidic patch on Dna2, which enables it to function with a sub-optimal amount of RPA, or to overcome DNA secondary structures. The trans-activity mode is not required for cell viability, but is necessary for effective double strand break (DSB) repair.

Highlights

Single-stranded DNA commonly occurs as intermediates in DNA metabolic pathways
Our study reveals a trans-acting element supported by a bimodal interface between Dna[2] and replication protein A (RPA), which allows Dna[2] within the Dna2–RPA–Single-stranded DNA (ssDNA) ensemble to interact with other RPA molecules and maintains its full activity when RPA becomes limiting or DNA secondary structures are encountered
We report that a separation-of-function mutant of DNA2—dna2-AC— which inactivates the trans-acting element, fully supports cell viability but causes a major defect in double strand break (DSB) repair, presumably due to a failure to resolve a critical recombination intermediate

Summary

Introduction

Single-stranded DNA (ssDNA) commonly occurs as intermediates in DNA metabolic pathways. Dna[2] removes RPA-coated long 5′-flap and allows Fen[1] nuclease to further complete lagging strand maturation[1]. This function of Dna[2] is essential for viability in yeast[6]. We combine biochemical, single-molecule, and genetic approaches to provide evidence that RPA, rather than acting passively[13], employs a gating mechanism to function as an active unit for the Dna[2] nuclease Within this framework, RPA allows its bound ssDNA to be released to Dna[2] within the ternary complex formed by Dna[2], RPA, and 5′-ssDNA. We report that a separation-of-function mutant of DNA2—dna2-AC— which inactivates the trans-acting element, fully supports cell viability but causes a major defect in DSB repair, presumably due to a failure to resolve a critical recombination intermediate

Methods

Results

Conclusion