Novel ssDNA-binding properties of SSB2 and SSB3 from Thermoanaerobacter tengcongensis

Abstract:

Abstract: [Objective] SSB2 and SSB3 are ssDNA-binding proteins of Thermoanaerobacter tengcongensis. This work aimed to disclose novel properties of both proteins. [Methods] We performed electrophoretic mobility shift assays (EMSAs) using oligonucleotides spanning the replication origin of T. tengcongensis and non-denaturing polyacrylamide gels. Western blotting assays were used to study the expression patterns of both proteins. [Results] SSB2 bound to 35-nt, 59-nt and 70-nt ssDNA spanning the replication origin and formed one, two or three DNA-protein complexes. The number of the SSB2-DNA complexes was determined by both the length of the ssDNA and the concentration of SSB2. SSB3 formed one more DNA-protein complex with 59-nt or 70-nt ssDNA in comparison with SSB2. Storage of the proteins at -70oC led to the disappearance of one SSB2-(70-nt) complex, or two SSB3-(59-nt) complexes or three SSB3-(70-nt) complexes in the EMSA, indicating the distinct loss of the SSBs’s conformations. Moreover, SSB2 and SSB3 displayed different expression patterns at variable incubation temperatures in vivo. [Conclusion] SSB2 and SSB3 could bind ssDNA with various conformations that were determined by the length of ssDNA, the concentration of the proteins, as well as the temperature of treatment. To our knowledge, this is the first disclosure of the characteristics of SSB2 and SSB3 on 35-70 nt oligonucleotides.