耐辐射球菌的单链DNA结合蛋白(Ssb)包含通过β发夹连接器连接的N-和C-末端寡核苷酸/寡糖结合(OB)折叠。要将功能角色分配给各个OB折叠,我们生成了三个Ssb变体:SsbN(无连接器的N端子),SsbNC(带连接器的N端子)和SsbC(C端子),每个都有一个OB折叠。SsbN和SsbNC均表现出弱的单链DNA(ssDNA)结合活性,与全长Ssb(SsbFL)相比。SsbC显示的ssDNA结合活性水平介于SsbFL和SsbN之间。SsbC和SsbFL主要以同二聚体形式存在,而SsbNC/SsbN形成不同的寡聚形式。体外,SsbNC或SsbN与SsbC形成显示增强的ssDNA结合活性的二元复合物。不像SsbFL,Ssb变体能够差异调节拓扑异构酶-I活性,但未能刺激DeinococcalRecA促进DNA链交换。结果表明,C-末端OB折叠主要负责ssDNA结合。N-末端OB折叠与ssDNA弱结合,但参与多聚化。
Single-stranded DNA binding protein (Ssb) of Deinococcus radiodurans comprises N- and C-terminal oligonucleotide/oligosaccharide binding (OB) folds connected by a beta hairpin connector. To assign functional roles to the individual OB folds, we generated three Ssb variants: SsbN (N-terminal without connector), SsbNC (N-terminal with connector) and SsbC (C-terminal), each harboring one OB fold. Both SsbN and SsbNC displayed weak single-stranded DNA (ssDNA) binding activity, compared to the full-length Ssb (SsbFL). The level of ssDNA binding activity displayed by SsbC was intermediate between SsbFL and SsbN. SsbC and SsbFL predominantly existed as homo-dimers while SsbNC/SsbN formed different oligomeric forms. In vitro, SsbNC or SsbN formed a binary complex with SsbC that displayed enhanced ssDNA binding activity. Unlike SsbFL, Ssb variants were able to differentially modulate topoisomerase-I activity, but failed to stimulate Deinococcal RecA-promoted DNA strand exchange. The results suggest that the C-terminal OB fold is primarily responsible for ssDNA binding. The N-terminal OB fold binds weakly to ssDNA but is involved in multimerization.