Direct Binding to Replication Protein A (RPA)-coated Single-stranded DNA Allows Recruitment of the ATR Activator TopBP1 to Sites of DNA Damage

Julyana Acevedo,Shan Yan,W Matthew Michael

doi:10.1074/jbc.m116.729194

Abstract

A critical event for the ability of cells to tolerate DNA damage and replication stress is activation of the ATR kinase. ATR activation is dependent on the BRCT (BRCA1 C terminus) repeat-containing protein TopBP1. Previous work has shown that recruitment of TopBP1 to sites of DNA damage and stalled replication forks is necessary for downstream events in ATR activation; however, the mechanism for this recruitment was not known. Here, we use protein binding assays and functional studies in Xenopus egg extracts to show that TopBP1 makes a direct interaction, via its BRCT2 domain, with RPA-coated single-stranded DNA. We identify a point mutant that abrogates this interaction and show that this mutant fails to accumulate at sites of DNA damage and that the mutant cannot activate ATR. These data thus supply a mechanism for how the critical ATR activator, TopBP1, senses DNA damage and stalled replication forks to initiate assembly of checkpoint signaling complexes.

Highlights

The maintenance of genome stability relies on faithful DNA replication and the ability of cells to suppress the mutagenic consequences of replication stress and DNA damage
Given that replication protein A (RPA)-single-stranded DNA (ssDNA) is a common feature of both stalled forks and double-strand breaks (DSBs) and that TopBP1 activates ATR in both contexts, we reasoned that interaction between TopBP1 and RPAssDNA is likely to be important for how TopBP1 is recruited to sites of damage
We present a new model for ATR activation at stalled forks. This model proposes that an early event in ATR activation is the direct interaction between TopBP1 and RPAssDNA at the stalled fork, via TopBP1’s BRCT2 domain

Summary

Introduction

The maintenance of genome stability relies on faithful DNA replication and the ability of cells to suppress the mutagenic consequences of replication stress and DNA damage. A TopBP1 point mutant (W265R) was identified that cannot accumulate at sites of replication stress, and in egg extract containing this mutant as the sole source of TopBP1, both Rad17 and 911 fail to associate with stalled replication forks and ATR is not activated [38].

Results

Conclusion