Characterization of single-stranded DNA-binding protein SsbB from Staphylococcus aureus: SsbB cannot stimulate PriA helicase. Issue 50 (7th August 2018) - Chen, Kuan-Lin
Abstract : Single-stranded DNA-binding proteins (SSBs) are essential to cells as they participate in DNA metabolic processes, such as DNA replication, repair, and recombination. Abstract : Single-stranded DNA-binding proteins (SSBs) are essential to cells as they participate in DNA metabolic processes, such as DNA replication, repair, and recombination. The functions of SSBs have been studied extensively in Escherichia coli . Unlike E. coli, which contains only one type of SSB (EcSSB), some bacteria have more than one paralogous SSB. In Staphylococcus aureus, three SSBs are found, namely, SsbA, SaSsbB, and SsbC. While EcSSB can significantly stimulate EcPriA helicase, SaSsbA does not affect the SaPriA activity. It remains unclear whether SsbBs can participate in the PriA-directed DNA replication restart process. In this study, we characterized the properties of SaSsbBs through structural and functional analyses. Crystal structure of SaSsbB determined at 2.9 Å resolution (PDB entry ;5YYU ) revealed four OB folds in the N-terminal DNA-binding domain. DNA binding analysis using EMSA showed that SaSsbB binds to ssDNA with greater affinity than SaSsbA does. Gene map analysis demonstrated that SAAV0835 encoding SaSsbB is flanked by unknown genes encoding hypothetical proteins, namely, putative Sipho_Gp157, ERF, and HNHc_6 gene products. Structure-based mutational analysis indicated that the four aromatic residues (Phe37, Phe48, Phe54, and Tyr82) in SaSsbB are at positions that structurally correspond to the important residues of EcSSB for binding to ssDNA and are also critical for SaSsbB to bind ssDNA. Similar to EcSSB and other SSBs such as SaSsbA and SaSsbC, SaSsbB also exhibited high thermostability. However, unlike EcSSB, which can stimulate EcPriA, SaSsbB did not affect the activity of SaPriA. Based on results in this study and previous works, we therefore established that SsbA and SsbB, as well as SsbC, do not stimulate PriA activity.