Dataset Information

SAAV2152 is a single-stranded DNA binding protein: the third SSB in Staphylococcus aureus.

ABSTRACT: Single-stranded DNA-binding proteins (SSBs) play crucial roles in DNA replication, repair, and recombination. Unlike E. coli, which contains only one type of SSB (EcSSB), some bacteria have two paralogous SSBs, namely, SsbA and SsbB. In this study, we found the third SSB-like protein in Staphylococcus aureus, SAAV2152, which was designated as SaSsbC. SaSsbC is a protein of 131 amino acids and shares 38%, 36%, and 33% sequence identity to SaSsbB, SaSsbA, and EcSSB, respectively. Gene map analysis showed that unlike the E. coli ssb gene, which is adjacent to uvrA gene, the S. aureus ssb gene SAAV2152 is flanked by the putative SceD, the putative YwpF, and fabZ genes. A homology model showed that SaSsbC consists of the classic oligonucleotide/oligosaccharide-binding fold at the N-terminus. At the C-terminus, SaSsbC did not exhibit sequence similarity to that of EcSSB. Electrophoretic mobility shift analysis showed that SaSsbC formed a single complex with ssDNA of different lengths. Mutational analysis revealed that Tyr36, Tyr47, Phe53, and Tyr81 in SaSsbC are at positions that structurally correspond to the important residues of EcSSB for binding to ssDNA and are also critical for SaSsbC to bind ssDNA. Unlike EcSSB, which can stimulate EcPriA, SaSsbC did not affect the activity of SaPriA. In addition, SaSsbA inhibitor 9-methyl-2,3,7-trihydroxy-6-fluorone (NSC5426) could inhibit the ssDNA-binding activity of SaSsbC with IC50 of 78 ?M. In conclusion, this study has identified and characterized SAAV2152 as a kind of SSB, and further research can directly focus on determining its actual physiological role in S. aureus.

SUBMITTER: Huang YH

PROVIDER: S-EPMC5945547 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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SAAV2152 is a single-stranded DNA binding protein: the third SSB in Staphylococcus aureus.

Huang Yen-Hua YH Huang Cheng-Yang CY

Oncotarget 20180205 29

Single-stranded DNA-binding proteins (SSBs) play crucial roles in DNA replication, repair, and recombination. Unlike E. coli, which contains only one type of SSB (EcSSB), some bacteria have two paralogous SSBs, namely, SsbA and SsbB. In this study, we found the third SSB-like protein in Staphylococcus aureus, SAAV2152, which was designated as SaSsbC. SaSsbC is a protein of 131 amino acids and shares 38%, 36%, and 33% sequence identity to SaSsbB, SaSsbA, and EcSSB, respectively. Gen ...[more]

PMID: 29755648

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Project description:Single-stranded DNA-binding protein (SSB) and PriA helicase play important roles in bacterial DNA replication restart process. The mechanism by which PriA helicase is bound and stimulated by SSB in Escherichia coli (Ec) has been established, but information on this process in Gram-positive bacteria are limited. We characterized the properties of SSB from Staphylococcus aureus (SaSsbA, a counterpart of EcSSB) and analyzed its interaction with SaPriA. The gel filtration chromatography analysis of purified SaSsbA showed a stable tetramer in solution. The crystal structure of SaSsbA determined at 1.82 Å resolution (PDB entry 5XGT) reveals that the classic oligonucleotide/oligosaccharide-binding folds are formed in the N-terminal DNA-binding domain, but the entire C-terminal domain is disordered. Unlike EcSSB, which can stimulate EcPriA via a physical interaction between EcPriA and the C-terminus of EcSSB (SSB-Ct), SaSsbA does not affect the activity of SaPriA. We also found that SaPriA can be bound by SaSsbA, but not by SaSsbA-Ct. Although no effect was found with SaSsbA, SaPriA can be significantly stimulated by the Gram-negative Klebsiella pneumoniae SSB (KpSSB). In addition, we found that the conserved SSB-Ct binding site of KpPriA (Trp82, Tyr86, Lys370, Arg697, and Gln701) is not present in SaPriA. Arg697 in KpPriA is known to play a critical role in altering the SSB35/SSB65 distribution, but this corresponding residue in SaPriA is Glu767 instead, which has an opposite charge to Arg. SaPriA E767R mutant was constructed and analyzed; however, it still cannot be stimulated by SaSsbA. Finally, we found that the conserved MDFDDDIPF motif in the Gram-negative bacterial SSB is DISDDDLPF in SaSsbA, i.e., F172 in EcSSB and F168 in KpSSB is S161 in SaSsbA, not F. When acting with SaSsbA S161F mutant, the activity of SaPriA was dramatically enhanced elevenfold. Overall, the conserved binding sites, both in EcPriA and EcSSB, are not present in SaPriA and SaSsbA, thereby no stimulation occurs. Our observations through structure-sequence comparison and mutational analyses indicate that the case of EcPriA-EcSSB is not applicable to SaPriA-SaSsbA because of inherent differences among the species.

Dataset Information

SAAV2152 is a single-stranded DNA binding protein: the third SSB in Staphylococcus aureus.

Publications

SAAV2152 is a single-stranded DNA binding protein: the third SSB in <i>Staphylococcus aureus</i>.

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