Cloning, expression, purification, crystallization and preliminary X-ray characterization of the full-length single-stranded DNA-binding protein from the hyperthermophilic bacteriumAquifex aeolicus

David J Clarke,Dmitriy Alexeev,Iain W Mcnae,Christopher G Northey,Lynsey A Mack,Lindsay Sawyer,Dominic J Campopiano

doi:10.1107/s0907444904020530

Abstract

Single-stranded DNA-binding (SSB) proteins stabilize single-stranded DNA, which is exposed by separation of the duplex during DNA replication, recombination and repair. The SSB protein from the hyperthermophile Aquifex aeolicus has been overexpressed in Escherichia coli, purified and characterized and crystals of the full-length protein (147 amino acids; M(r) 17 131.20) have been grown by vapour diffusion from ammonium sulfate pH 7.5 in both the absence and presence of ssDNA [dT(pT)(68)]. All crystals diffract to around 2.9 A resolution and those without bound DNA (native) belong to space group P2(1), with two tetramers in the asymmetric unit and unit-cell parameters a = 80.97, b = 73.40, c = 109.76 A, beta = 95.11 degrees . Crystals containing DNA have unit-cell parameters a = 108.65, b = 108.51, c = 113.24 A and could belong to three closely related space groups (I222, I2(1)2(1)2(1) or I4(1)) with one tetramer in the asymmetric unit. Electrospray mass spectrometry of the crystals confirmed that the protein was intact. Molecular replacement with a truncated E. coli SSB structure has revealed the position of the molecules in the unit cell and refinement of both native and DNA-bound forms is under way.

Highlights

Single-stranded DNA-binding (SSB) proteins have been shown to play an essential role in many aspects of DNA metabolism (Chase & Williams, 1986)
It is clear from the sequence alignment of the SSB proteins that the A. aeolicus and the E. coli proteins are more closely related to each other than either is to S. solfataricus SSB (Fig. 1)
This is borne out by the fact that a molecular-replacement solution using the E. coli structure was obtained relatively whilst no satisfactory solution could be obtained with the S. solfataricus structure

Summary

Introduction

Single-stranded DNA-binding (SSB) proteins have been shown to play an essential role in many aspects of DNA metabolism (Chase & Williams, 1986). Bacterial SSB proteins form homotetramers, with each subunit containing one DNA-binding domain (Raghunathan et al, 1997). The C-terminal domain of bacterial SSB proteins exhibits low sequence homology across species, with the exception of the terminal six residues, which form a highly conserved negatively charged DDDIPF motif This motif is essential for the function of Escherichia coli SSB protein in vivo (Curth et al, 1996) and has been shown to interact directly with the 3H±5H ssDNA-degrading exonuclease I (Genschel et al, 2000). Theoretical weight of full-length A. aeolicus SSB protein (17 131.2 Da)

Crystallization

Data collection and processing

Discussion