Abstract
Single stranded DNA (ssDNA) binding protein (SSB) plays essential roles in many processes related to DNA metabolism such as DNA replication, repair, and homologous genetic recombination. The HP1245 gene was annotated as SSB in Helicobacter pylori strain 26695. However, there are no functional or structured studies for this SSB up to now. The full length (179 residues) HP1245, a C-terminal truncated HP1245 containing 134 resides (tmHP1245 (1~134)), and four HP1245 mutants each containing 134 residues with a specific site mutant (F37A, F50A, F56A and W84A) were individually cloned into pQE30 vector and expressed in E. coli SG13009 previously in our lab. Applied basic gene cloning techniques and available plasmids, the gene containing full length HP1245 SSB with a point mutation on F37A, F50A, F56A or W84A was separately constructed and expressed in pQE30 containing E. coli SG13009 system. In addition, EcoSSB containing expression vector in E. coli SG13009 system was also prepared in this study. The binding activity between single stranded DNA and any one of the above mentioned fresh prepared HP1245 proteins (full length wild type, full length point mutants and C-terminal truncated mutant) were measured by means of (a) fluorescence titration (fixed amount of SSB plus fragmented calf thymus ss-DNA, or 600-1200 bp), (b) Electrophoresis mobility shift assay (EMSA) (SSB plus fixed amount of M13mp18 ssDNA) and (c) SPR (biotin-labeled d(T)35 ssDNA) in this study. The results of the fluorescence titration from the measurement of the tryptophan quenching due to the ssDNA binding to SSB provided one useful parameter, binding site sizes of nucleotides or Napp to wrap around (or cover) a SSB tetramer. Under high salt condition at 300 mM NaCl, the Napp for the full length HP1245 SSB was 35 + 2 nucleotides/tetramer, shorter than that for EcoSSB (61 + 4 NT/tetramer). The tmHP1245 owned the shortest Napp (30 + 1 NT/tetramer) among the various HP1245 SSB mutants used in this study. Similar results were also observed from SSB binding to ssDNA at low salt 10 mM NaCl condition, suggesting that the C-terminal of HP1245 SSB should play a role on ssDNA binding.On the other hand, EMSA results on retardation of the single stranded M13mp18 plasmid DNA migration on DNA agarose gel during SSB binding showed that about 330~439 molecules of full length HP1245 tetramer would saturate to bind one molecule of M13mp18 at high or low salt. Strong positive co-operativity showed in wild type HP1245 SSB-M13 complexes only at low salt condition. More tm-HP1245 tetramers were required to saturate the binding of single M13 molecule, indicating the C-terminal region of HP1245 affected the retardation of EMSA.The interaction of a series SSB with ssDNA has been further measured in real time by using a surface plasmon-resonance (SPR) and biosensor chip. Wild type HP1245 SSB was first applied onto the sensor chip surface that was pre-coated with Au, MUA, EDC/NHS, Streptoavidin and 5’-Biotinyl-poly(dT)35 in order. SPR measure at different conditions including: strpetavidin immobilization buffer pH value (Figure 11), 5’ biotinyl-poly(dT)35 capacity (Figure 12), flow rate of kinetic experiments (Figure13), regeneration buffer (Figure 14), HP1245 protein concentration and association time (Figure 16) were examined to obtain optimal conditions for further DNA binding experiments for each of above mentioned various HP1245 SSB. Response unit (RU) data from SPR after processing software program, BIAevaluation version 4.1 were transformed into useful parameters, such as ka, kd, KA, KD etc. to describe the SSB and ssDNA binding affinity.The KD of wild type HP1245 SSB binding poly dT 35mer was 0.16 nM in using BiacoreX (Figure 16A) and 0.1 nM in using Biacore3000 (Figure 16B), although different association time was used, 2 min for the former and 5 min for the latter. Higher KD (1.64 nM, about 9.9-fold, Figure 18) was obtained for C-terminal tm-HP1245 (1~134) in comparison with that from full-length HP1245 SSB to bind ssDNA. This result again emphasized that C-terminal of HP1245 was important for ssDNA binding. The importance of the C-terminal region on HP1245 was confirmed in results from fluorescence titration, EMSA and SPR measurement in this study.KD value for the binding of SSB to ssDNA from the lowest to highest is 0.16 nM for full length wild type HP1245 SSB, 1.6 nM (10-fold) for F37A mutant (Figure 19), 1.64 nM (10-fold) for tm-HP1245 (1~134) (Figure 18), 2.3 nM (14.1-fold) for W84A mutant (Figure 19), 3.06 nM (18.4-fold) for F50A mutant (Figure 19) and 3.12 nM (18.6-fold) for F56A mutant (Figure 20). These results suggested that the mutation of F37, F50, F56 or W84 in HP1245 SSB affected its binding to ssDNA, resulting in less KA (more binding affinity between SSB and ssDNA) or more KD (less dissociation for SSB-ssDNA complex) than that for wild type HP1245 SSB.Thus, SPR analysis was the most convenient method to examine the binding between ssDNA and different HP1245 SSB. It demonstrated that HP1245 SSB binds single stranded DNA with high affinity, by involving a tryptophan residue W84, and 3 phenylalanines F37, F50 and F56. Either SPR, fluorescence titration or EMSA could be used to distinguish different KD between tm-HP1245 SSB and wild type HP1245 SSB for ssDNA binding, higher KD (10-fold) in the former than that in the latter. This confirmed that the C-terminal region of HP1245 SSB was also important to bind ssDNA.