The Function Of Arginine Finger And HRDC Domain Of Bacillus Subtilis Recq Helicase

RecQ helicaeses are one of the most important macromolecules in the process of molecular metabolism. They play essential roles in maintaining the stability of the genetic materials in cells by facilitating cellular processes including genome replication, DNA repair, recombination, transcription and telomere maintenance. Many helicases have been found, but they have a certain homology and similarity in the amino acid sequence. In this paper, we focus on the structures and functions of Bacillus subtilis RecQ helicases. We studied the relationship between the structure and biochemical activity of them, and revealed the molecular mechanism of the Bacillus subtilis RecQ helicases. Our previous work showed that there were two isoforms of RecQ helicases in Bacillus subtilis, the typical structural differences between these two RecQ helicases were that one contains a C-terminal amino acid structure called HRDC region, while was missing in other member. These two proteins are ideal models for study the HRDC domain. The arginine residues of Bacillus subtilis RecQ played important roles in ATP hydrolyzation and binding activities. The DNA corresponding to the coding sequence of the Bacillus subtilis RecQ helicase gene was respectively amplified from the chromosome DNA of Bacillus subtilis168and the B.subtilis RecQ L-pET15b by PCR, one is about1.5kb and the other is1.8kb. The arginine residues of Bacillus subtilis RecQ were mutated separately or simultaneously to alanine residues by overlapping PCR method, then wild type and mutants were subcloned into the expression vector pET24a(+). The recombinant proteins were induced with IPTG to express in E.coli BL21(DE3). All of the proteins obtained in vitro were with above90%purity and good solubility, and then the biochemical activity of wild type and mutants were detected. The results showed that Bacillus subtilis RecQ and mutants had DNA-dependent ATPase activity in concentration-dependent manner. However, the ATP hydrolysis activities of mutants were significantly reduced compared with the wild RecQ. The consequence state that the two arginine residues took an important part in interacting RecQ helicaese with ATP. Orthovanadate (Vi) is widely used to probe the catalytic mechanism of nucleotide-5’-triphosphotase. Not all ATPases are inhibited by Vi. If the ATPase activity of a protein is efficiently inhibited by Vi, it suggests, although does not prove, that the catalytic site of the protein may share structural features with other ATPases that have an arginine finger. The findings indicated that both B.subtilis RecQ S R322and B. subtilis RecQ L R323played roles as arginine fingers. To observe the interaction between RecQ helicase and DNA, we used EMSA and fluorescence polarization to detect this activity. However, it is not sensitive by EMSA, so we use the fluorescence polarization to detect the DNA binding activity. Results showed that Bacillus subtilis RecQ helicase had DNA-dependent activity in concentration-dependent manner. The mutants may affect the ability of DNA binding, the arginine residue could increase DNA binding with RecQ. The article also measured the DNA helicase activity, annealing activity and ATP binding activity by the FRET. The activity of DNA helicase and ATP binding were significantly reduced, which indicated these arginine residues could increased the activity of DNA unwinding and ATP binding. But the mutants did not affect severely about the annealing. In addition, the paper truncated the HRDC region of Bacillus subtilis RecQ L. We found that HRDC region also could play a role in the biological activity including DNA binding, ATPase activities. These results are conducive to study the structures and functions of other members of the RecQ family helicases.