Tetramerization and Single-stranded DNA Binding Properties of Native and Mutated Forms of Murine Mitochondrial Single-stranded DNA-binding Proteins

Kang Li,R Sanders Williams

doi:10.1074/jbc.272.13.8686

Abstract

We examined previously unexplored aspects of the tetramerization and single-stranded DNA (ssDNA) binding properties of native, precursor, and mutated forms of mitochondrial ssDNA-binding protein (mtSSB) from a mammalian organism (mouse). Tetramic forms of mtSSB reassemble spontaneously after thermal denaturation and undergo subunit exchange. Binding of mtSSB to ssDNA as a function of protein concentration is nonlinear, suggesting a concentration-dependent transition in intrinsic binding affinity and in the topology of the DNA-protein complex. The cleavable presequence at the amino terminus of the precursor form of mtSSB does not disrupt tetramer formation but has a specific inhibitory effect on DNA binding that is not seen in a fusion protein that substitutes a bulkier peptide moiety in this position. Mutated forms of mtSSB bearing amino acid substitutions at highly conserved amino acid positions exhibit subtle or severe defects in ssDNA binding activity and/or tetramerization, even when assembled into heterotetramers in combination with wild-type mtSSB monomers. These experiments provide new insights into structural and functional properties of mammalian mtSSB and have implications for the pathogenesis of human diseases resulting from defects in mtDNA replication.

Highlights

Single-stranded DNA-binding proteins (SSBs)1 are involved in DNA replication, repair, and recombination in prokaryotic organisms and in both nuclei and mitochondria of eukaryotes
A murine mitochondrial SSB (mtSSB) cDNA was cloned by PCR amplification, and several forms of recombinant mtSSB were purified following expression in bacteria: the mature native protein; an epitope-tagged mtSSB; an unprocessed mtSSB bearing the presequence required for mitochondrial import; a fusion protein in which glutathione S-transferase (GST) is linked to the amino terminus of mtSSB; and mtSSBbearing amino acid substitutions at each of four highly conserved amino acid positions
Using a bacterial expression system, we purified several different forms of mtSSB (Fig. 1B): wild-type mature protein (WT), wild-type precursor protein (WP), wild-type mature protein with a His-tag at the carboxyl terminus (WH), and wild-type mature protein fused to GST at the amino terminus

Summary

Introduction

Single-stranded DNA-binding proteins (SSBs) are involved in DNA replication, repair, and recombination in prokaryotic organisms and in both nuclei and mitochondria of eukaryotes. Occurring point mutations in E. coli SSBs cause pleiotrophic defects in replication, repair of UV-damaged DNA, and recombination (8 –11). The amino acid sequence of mitochondrial SSB (mtSSB) has been determined previously in several species, including human, rat, Xenopus, and Saccharomyces cerevisiae [12,13,14,15,16]. These eukaryotic proteins include a number of conserved residues shared with E. coli SSBs in their amino-terminal regions but are divergent otherwise. We used gel mobility shift assays, equilibrium sucrose gradient centrifugation, and immunoprecipitation to characterize the ssDNA binding properties and the dissociation and re-assembly of mtSSB homo- and heterotetramers following thermal denaturation

Methods

Results

Conclusion