Screening The Interacting Proteins Of PIF1by The Yeast Two-hybrid

Helicase are enzymes which can catalyze the unwinding of the DNA duplex, which playimportant roles in almost all the nucleic acid metabolism, including DNA replication,transcription, recombination and DNA repair. There exit some highly conserved amino acidsequences in the primary structure of helicases, which is called helicase motifs.PIF1helicases are5’-3’ helicases, very conserved from yeast to human. The yeast Pif1participate in DNA repair, regulation of telomere length, DNA replication and heritablestability maintaining. Very little is known about human DNA helicase. In our previous study,by bioinformatics analysis, we found that the N-terminus of human PIF1is also veryconserved, so we defined it PINT domain(PIF1N-terminal，domain). By biochemical analysis,we further found PINT domain confered the annealing activity and played important roles inregulation the PIF1helicase activity.The purpose of this research is to screen the upstream signal molecular and theinteraction target proteins by yeast two-hybrid system.We used the PINT domain and helicase domain of human PIF1helicase as bait to screena human HeLa library in order to find the protein interacting with individual domains. Baitfragments were amplified by PCR and inserted into pGB, a yeast expression plasmid. ThepGB-Bait plasmids were verified by DNA sequdence and transformed into yeast Y190. Usingβ-galactosidase chromogenic assy to check whether the bait proteins can be self-activated.Then human HeLa library was transformed into yeast strains which can stably express baitprotein. The positive colonies were screened with defected inflat panel (SD/-Leu-Trp-His+3AT). The positive plasmid and pGB-Bait were co-transformed into Y190to further confirmthe positive clones. Finally, the library plasmids of positive clones were analyzed bybioinformatics.Ultimately,15positive clones were obtained using pGB-PIF1N bait protein to screen thehuman Hela library. And by sequencing and analyzing the positive clones, three positivegenes were identified: CCNDBP1, OTUD5, CAP1. However, no positive clone was obtainedusing bait pGB-PIF1C to screen human HeLa library.Interacting proteins with PINT domain were identified by yeast two-hybrid assay，which provides us some new clues for further research of PIF1in the role of apoptosis anddamage restoration.