The Mutagenesis Of Phosphorylation Modified Sites Of POL5 And Its Effect On Pol5 Protein Expression In Schizosaccharomyces Pombe

Objective:Pol5 protein is essential for cell growth. Pol5 protein is strict conservation from human beings to the yeast.In human beings the homologs of Pol5 protein is MYBBP1A(Myb-binding protein 1A),which is a nucleolar protein implicated in nucleolar stress response and carcinogenesis.Some previous studys reported that the essential function of Pol5 protein is r DNA transcription. In our previous study, we found that it might be a potential substrate of Eso1 protein. Additionally,we found that the 742,743 serine sites of Pol5 protein is phosphorylation modified.However,the function of the 742,743 serine sites of Pol5 protein phosphorylation modified contribute to cell growth and protein function remains elusive. In our study,we cunstructed the mutations of phosphorylation modified sites of POL5,which tagged with HA signal and analysis its effect on Pol5 protein expression in Schizosaccharomyces pombe. Schizosaccharomyces pombe strains use to study the function of phosphorylation modified sites of POL5 may be constructed.Methods:In the previous study,we constructed pdbletpol5 HA and p Clone Natpol5 plasmid,which contain the same double restrction enzyme recognition sites.We got POL5 HA from pdbletpol5 HA plasmid and p Clone Nat from p Clone Natpol5 plasmid.Then,we got the new plasmid by T4 DNA ligation repair the DNA fragments.The new plasmid p Clone Natpol5 HA contains the POL5 gene sequence,HA-signal peptide gene sequence, Nat-Tagged gene sequence,Anti-Amp gene sequence and stop code.We got the linear plasmid DNA after digested with the Afe1.Then,we transformed the linear plasmid DNA into the S.pombe.After sequencing and western blot,we conformed it is the right plasmid.We intergrated imitatation phosphorylation and dephosphorylation amino acid sequence into the p Clone Natpol5 HA by sit-directed mutagenesis technology.Then,we observed the expression of the pol5 and the growth of cell.Results:1. The POL5 protein can express normally in S.pombe afer tagged with HA-signal.2. We got the Pol5S742AS743 A imitatation dephosphorylation mutations strain successfully.3. We got the Pol5S742DS743 D imitatation phosphorylation mutations strain successfully.4. Nor intergrate imitation phosphorylation or dephosphorylation amino acid sequence into the pol5 gene can effect the expression of Pol5 protein.Conclusions: The strains mutagenesis in the 742,743 serine phosphorylation modified sites of POL5 can survive.Thus,those mutant strains may provide a successful model for our next study th the function of Pol5 protein phosphorylation modifying.The effect of the mutagenesis in pol5 can not lead to the change of the protein expression,consider that we may purify the phosphorylation protein in the following study conveniently. Experimental analysis of pol5 is likely to shed light on the function of pol5 phosphorylation.