Crystal structure of DnaT84-153-dT10 ssDNA complex reveals a novel single-stranded DNA binding mode.

Zheng Liu,Maikun Teng,Gang Cai,Xu Li,Liwen Niu,Peng Chen,Xuejuan Wang

doi:10.1093/nar/gku633

Abstract

DnaT is a primosomal protein that is required for the stalled replication fork restart in Escherichia coli. As an adapter, DnaT mediates the PriA-PriB-ssDNA ternary complex and the DnaB/C complex. However, the fundamental function of DnaT during PriA-dependent primosome assembly is still a black box. Here, we report the 2.83 Å DnaT84–153-dT10 ssDNA complex structure, which reveals a novel three-helix bundle single-stranded DNA binding mode. Based on binding assays and negative-staining electron microscopy results, we found that DnaT can bind to phiX 174 ssDNA to form nucleoprotein filaments for the first time, which indicates that DnaT might function as a scaffold protein during the PriA-dependent primosome assembly. In combination with biochemical analysis, we propose a cooperative mechanism for the binding of DnaT to ssDNA and a possible model for the assembly of PriA-PriB-ssDNA-DnaT complex that sheds light on the function of DnaT during the primosome assembly and stalled replication fork restart. This report presents the first structure of the DnaT C-terminal complex with ssDNA and a novel model that explains the interactions between the three-helix bundle and ssDNA.

Highlights

A faithful and reliable transmission of genetic material requires precise coordination and regulation of chromosome replication [1,2]
Because it has been reported that both single-stranded DNA-binding proteins (SSBs) and PriB can act as sequence-independent ssDNA chaperones [41], which do not limit the conformation of the bound ssDNA to the extent that is observed for other known DNA-binding proteins [21], and because we did perform the experiments to identify the ability of DnaT binding to longer ssDNA, we modelled the longer helical filament shape oligomerised DnaT84–153 –ssDNA according to crystal packing (Figure 5B)
The structural and biochemical characterisation on E. coli DnaT that we reported here revealed a novel ssDNA-binding mode that is distinct from all of those mentioned

Summary

Introduction

A faithful and reliable transmission of genetic material requires precise coordination and regulation of chromosome replication [1,2]. This process requires the assembly of a replication complex, a primosome, at the origin [3]. DnaA recognition and binding to oriC recruits DnaB helicase with the help of the helicase loader DnaC and coordinates the DnaB helicase and the primase DnaG functions [1,11,12,13,14]. The PriA binds to n -pas while assisted by the auxiliary PriB, PriC, DnaT, to recruit the DnaB, DnaC and DnaG [16]. The two distinct primosomes share the same helicase DnaB and DnaG primase systems

Methods

Results

Conclusion