C-terminal domain swapping of SSB changes the size of the ssDNA binding site.

Yen-Hua Huang,Cheng-Yang Huang

doi:10.1155/2014/573936

Abstract

Single-stranded DNA-binding protein (SSB) plays an important role in DNA metabolism, including DNA replication, repair, and recombination, and is therefore essential for cell survival. Bacterial SSB consists of an N-terminal ssDNA-binding/oligomerization domain and a flexible C-terminal protein-protein interaction domain. We characterized the ssDNA-binding properties of Klebsiella pneumoniae SSB (KpSSB), Salmonella enterica Serovar Typhimurium LT2 SSB (StSSB), Pseudomonas aeruginosa PAO1 SSB (PaSSB), and two chimeric KpSSB proteins, namely, KpSSBnStSSBc and KpSSBnPaSSBc. The C-terminal domain of StSSB or PaSSB was exchanged with that of KpSSB through protein chimeragenesis. By using the electrophoretic mobility shift assay, we characterized the stoichiometry of KpSSB, StSSB, PaSSB, KpSSBnStSSBc, and KpSSBnPaSSBc, complexed with a series of ssDNA homopolymers. The binding site sizes were determined to be 26 ± 2, 21 ± 2, 29 ± 2, 21 ± 2, and 29 ± 2 nucleotides (nt), respectively. Comparison of the binding site sizes of KpSSB, KpSSBnStSSBc, and KpSSBnPaSSBc showed that the C-terminal domain swapping of SSB changes the size of the binding site. Our observations suggest that not only the conserved N-terminal domain but also the C-terminal domain of SSB is an important determinant for ssDNA binding.

Highlights

Single-stranded DNA-binding protein (SSB) binds to single-stranded DNA and is known to have important functions in the DNA metabolic processes, such as DNA replication, repair, and recombination of both prokaryotes and eukaryotes [1,2,3,4]
Based on the nucleotide sequence found, using a database search through the National Center for Biotechnology Information (NCBI), we predicted that Klebsiella pneumoniae SSB (KpSSB), Serovar Typhimurium LT2 SSB (StSSB), and PAO1 SSB (PaSSB) monomer proteins have lengths of 174, 176, and 165 amino acid residues, respectively
The important motif in the C-terminal tail of E. coli SSB, DDDIPF residues, is conserved in KpSSB, StSSB, and PaSSB. By contrast to those motifs, the residues found in the glycinerich hinge of E. coli SSB are not conserved in KpSSB, StSSB, and PaSSB (Figure 2)

Summary

Introduction

Single-stranded DNA-binding protein (SSB) binds to single-stranded DNA (ssDNA) and is known to have important functions in the DNA metabolic processes, such as DNA replication, repair, and recombination of both prokaryotes and eukaryotes [1,2,3,4]. During these reactions, SSB binds to and protects susceptible ssDNA from nucleolytic digestion and chemical attacks and prevents secondary structure formation [5].

Results

Discussion

Conclusion