C-Terminal Domain Swapping of SSB Changes the Size of the ssDNA Binding Site
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-prot...
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doaj-611c08cb0f5040f5b78a51223d67dc9c2020-11-24T22:32:07ZengHindawi LimitedBioMed Research International2314-61332314-61412014-01-01201410.1155/2014/573936573936C-Terminal Domain Swapping of SSB Changes the Size of the ssDNA Binding SiteYen-Hua Huang0Cheng-Yang Huang1School of Biomedical Sciences, Chung Shan Medical University, No.110, Sec.1, Chien-Kuo N. Rd., Taichung City, TaiwanSchool of Biomedical Sciences, Chung Shan Medical University, No.110, Sec.1, Chien-Kuo N. Rd., Taichung City, TaiwanSingle-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.http://dx.doi.org/10.1155/2014/573936 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Yen-Hua Huang Cheng-Yang Huang |
spellingShingle |
Yen-Hua Huang Cheng-Yang Huang C-Terminal Domain Swapping of SSB Changes the Size of the ssDNA Binding Site BioMed Research International |
author_facet |
Yen-Hua Huang Cheng-Yang Huang |
author_sort |
Yen-Hua Huang |
title |
C-Terminal Domain Swapping of SSB Changes the Size of the ssDNA Binding Site |
title_short |
C-Terminal Domain Swapping of SSB Changes the Size of the ssDNA Binding Site |
title_full |
C-Terminal Domain Swapping of SSB Changes the Size of the ssDNA Binding Site |
title_fullStr |
C-Terminal Domain Swapping of SSB Changes the Size of the ssDNA Binding Site |
title_full_unstemmed |
C-Terminal Domain Swapping of SSB Changes the Size of the ssDNA Binding Site |
title_sort |
c-terminal domain swapping of ssb changes the size of the ssdna binding site |
publisher |
Hindawi Limited |
series |
BioMed Research International |
issn |
2314-6133 2314-6141 |
publishDate |
2014-01-01 |
description |
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. |
url |
http://dx.doi.org/10.1155/2014/573936 |
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