Structures of GapR reveal a central channel which could accommodate B-DNA

Michael J Tarry,James A Taylor,Christoph Harmel,Gregory T Marczynski,T Martin Schmeing

doi:10.1038/s41598-019-52964-2

Abstract

GapR is a nucleoid-associated protein required for the cell cycle of Caulobacter cresentus. We have determined new crystal structures of GapR to high resolution. As in a recently published structure, a GapR monomer folds into one long N-terminal α helix and two shorter α helices, and assembles into a tetrameric ring with a closed, positively charged, central channel. In contrast to the conclusions drawn from the published structures, we observe that the central channel of the tetramer presented here could freely accommodate B-DNA. Mutation of six conserved lysine residues lining the cavity and electrophoretic mobility gel shift experiments confirmed their role in DNA binding and the channel as the site of DNA binding. Although present in our crystals, DNA could not be observed in the electron density maps, suggesting that DNA binding is non-specific, which could be important for tetramer-ring translocation along the chromosome. In conjunction with previous GapR structures we propose a model for DNA binding and translocation that explains key published observations on GapR and its biological functions.

Highlights

In prokaryotes, control of DNA replication and related processes for cell division are aided by a family of DNA-binding proteins known as nucleoid-associated proteins (NAPs)[1]
In this paper we present three crystal structures of GapR from C. crescentus, crystallised in the presence of double-stranded DNA with a sequence from the origin of chromosome replication
We propose that our crystals capture a physiologically-relevant GapR structure distinct from and complementary to the physiologically-relevant structures captured by Guo et al we combine these structural data and present an updated model for DNA binding and translocation which more fully accounts for the observed dynamics of GapR binding

Summary

Introduction

Control of DNA replication and related processes for cell division are aided by a family of DNA-binding proteins known as nucleoid-associated proteins (NAPs)[1]. GapR is an essential NAP in Caulobacter crescentus involved in DNA replication, chromosome segregation and cell division[2,3,4]. In the structure of GapR determined in presence of DNA, GapR is seen to be a tetramer, with the C-terminal α helix of each monomer reorganized into two shorter helices, which allows GapR to encircle the DNA. GapR is proposed to associate with positively supercoiled chromosomal DNA5, which is often found ahead of the replication fork and RNA polymerase[6]. In this paper we present three crystal structures of GapR from C. crescentus, crystallised in the presence of double-stranded DNA with a sequence from the origin of chromosome replication. We propose that our crystals capture a physiologically-relevant GapR structure distinct from and complementary to the physiologically-relevant structures captured by Guo et al we combine these structural data and present an updated model for DNA binding and translocation which more fully accounts for the observed dynamics of GapR binding

Methods

Results

Conclusion