Characterization of a Single-Stranded DNA-Binding Protein from Pseudomonas aeruginosa PAO1

Abstract

Single-stranded DNA-binding protein (SSB) plays an important role in DNA metabolism, such as in DNA replication, repair, and recombination, and is essential for cell survival. We characterized the single-stranded DNA (ssDNA)-binding properties of Pseudomonas aeruginosa PAO1 SSB (PaSSB) by using fluorescence quenching measurements and electrophoretic mobility shift analysis (EMSA). Analysis of purified PaSSB by gel filtration chromatography revealed a stable tetramer in solution. In fluorescence titrations, PaSSB bound 22-32 nucleotides (nt) per tetramer depending on salt concentration. Using EMSA, we characterized the stoichiometry of PaSSB complexed with a series of ssDNA homopolymers, and the size of the binding site was determined to be 29 ± 1 nt. Furthermore, EMSA results indicated that the dissociation constants of PaSSB for the first tetramer were less than those for the second tetramer. On the basis of these biophysical analyses, the ssDNA binding mode of PaSSB is expected to be noncooperative.

C-Terminal Domain Swapping of SSB Changes the Size of the ssDNA Binding Site

“…The KpSSB [40], StSSB [43], and PaSSB [42] expression plasmids were constructed by the protocols described previously, with minor modification, to avoid having a His tag fused with the gene product. A fragment containing the coding sequence of KpSSB ( KPN04446 ), StSSB ( STM4256 ), and PaSSB ( PA4232 ) (with the stop codon) was directly amplified by PCR by using the genomic DNA of K. pneumoniae subsp.…”

“…The recombinant SSBs were expressed using the protocol described previously [9, 40, 42, 43, 56–60]. Purification of these recombinant SSBs was carried out as described previously with the following modifications [61, 62].…”

“…For example, differences are found among the binding site sizes of Methanococcus jannaschii SSB [51], the Gonococcal Genetic Island-encoded SSB from Neisseria gonorrhoeae [39], the thermostable Thermotoga maritima and Thermotoga neapolitana SSBs [32], and the psychrophilic bacterial SSBs [37]. In addition, the (SSB) 35 - and (SSB) 65 -binding modes are not found in some SSBs [32, 39, 42]. …”

“…Previously, we have examined the electrophoretic mobility shift patterns of a His-tagged Klebsiella pneumoniae SSB (KpSSB) [40], a His-tagged Salmonella enterica serovar Typhimurium LT2 SSB (StSSB) [43], and a His-tagged Pseudomonas aeruginosa PAO1 SSB (PaSSB) [42] bound to different lengths of ssDNA. We also determined their corresponding binding site sizes, that is, 26, 22, and 29 nt per tetramer, respectively.…”

“…KpSSB, StSSB, and PaSSB are similar proteins whose N-terminal ssDNA-binding domains are almost identical, except for different ssDNA-binding site sizes [40, 42, 43]. Thus, we should assess whether the glycine-rich hinge, which is not conserved among SSBs, is involved in the determination of the binding site size of SSB.…”