Architecture and ssDNA interaction of the Timeless-Tipin-RPA complex.

Justine Witosch,Eva Wolf,Naoko Mizuno

doi:10.1093/nar/gku960

Abstract

The Timeless-Tipin (Tim-Tipin) complex, also referred to as the fork protection complex, is involved in coordination of DNA replication. Tim-Tipin is suggested to be recruited to replication forks via Replication Protein A (RPA) but details of the interaction are unknown. Here, using cryo-EM and biochemical methods, we characterized complex formation of Tim-Tipin, RPA and single-stranded DNA (ssDNA). Tim-Tipin and RPA form a 258 kDa complex with a 1:1:1 stoichiometry. The cryo-EM 3D reconstruction revealed a globular architecture of the Tim-Tipin-RPA complex with a ring-like and a U-shaped domain covered by a RPA lid. Interestingly, RPA in the complex adopts a horse shoe-like shape resembling its conformation in the presence of long ssDNA (>30 nucleotides). Furthermore, the recruitment of the Tim-Tipin-RPA complex to ssDNA is modulated by the RPA conformation and requires RPA to be in the more compact 30 nt ssDNA binding mode. The dynamic formation and disruption of the Tim-Tipin-RPA-ssDNA complex implicates the RPA-based recruitment of Tim-Tipin to the replication fork.

Highlights

DNA replication relies on the coordinated action of replisome components including a helicase, a primase, replicative polymerases and regulatory proteins [1]
Replication Protein A (RPA) in the Tim-Tim interacting protein (Tipin)-RPA complex employs a compact conformation similar to the previously characterized complex of RPA bound to single-stranded DNA (ssDNA) [19,20]
Our study shows that Tim-Tipin locks RPA in a conformation resembling to its 30 nt binding mode under DNA-free conditions

Summary

Introduction

DNA replication relies on the coordinated action of replisome components including a helicase, a primase, replicative polymerases and regulatory proteins [1]. Regulatory components, such as the fork protection complex (FPC), ensure correct duplication of the genome [2]. The depletion of Tim-Tipin causes uncoupling of polymerase-helicase, resulting in the accumulation of unwound single-strand DNA (ssDNA) covered by replication protein A (RPA) [11,12]. These findings lead to the hypothesis that Tim-Tipin may physically stabilize the replisome by bridging the helicase and polymerase [4]

Methods

Results

Conclusion