Both OB folds of single-stranded DNA-binding protein are essential for its ssDNA binding activity in Deinococcus radiodurans.
The single-stranded DNA-binding proteins are crucial in all kinds of DNA metabolic processes. Deinococcus SSB-like proteins are homodimers in nature and contain two OB folds per monomer, in contrast to other bacterial SSB proteins that are functionally active as homotetramers. We generated four truncated variants of DraSSB protein, based on its crystal structure (PDB code: 1SE8). Gel filtration showed that DraSSB, DSCT (lack C-tail) and DSCC (lack C-tail and C-terminal OB) were mostly homodimers, and DSN (lack N-terminal OB) and DSNC (lack N-terminal OB and connector) were mostly monomers. The gel filtration supported the hypothesis that the N-terminal domain played a predominant role in dimerisation. Biochemical characterization was used to determine the role of each OB fold in DNA binding, by EMSA and FRET. EMSA results suggested that binding of DraSSB to ssDNA substrate needed both N- and C-terminal OB-folds, and also their interaction to achieve optimum DNA binding. DSCT might possess two ssDNA binding modes compared with DraSSB. The C-terminal tail was not essential for binding of ssDNA substrates. The C-terminal OB-fold had the ability to bind to the bubble structure. Furthermore, the FRET results for DSCT verified the hypothesis that DSCT showed two different binding modes for ssDNA, similarly to EcoSSB.
Hua
Xiaoting
X
Laboratory for Nuclear-Agricultural Sciences of Chinese Ministry of Agriculture and Zhejiang Province, Institute of Nuclear-Agricultural Sciences, Zhejiang University, 310029, Hangzhou, China.
Wang
Chao
C
Zhao
Ye
Y
Wang
Hu
H
Huang
Lifen
L
Xu
Guangzhi
G
Li
Mingfeng
M
Wang
Yuan
Y
Tian
Bing
B
Hua
Yuejin
Y
D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D034301:Deinococcus / Q000235:genetics* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D031541:Fluorescence Resonance Energy Transfer; D014644:Genetic Variation; D017510:Protein Folding; D016415:Sequence Alignment
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins