The Study On Molecule Mechanism Of Candida Telomerase Extend Telomeric DNA

Telomerase, consisting of a protein reverse transcriptase?TERT? and an RNA subunit?TR?, is a specialized reverse transcriptase?RT? that maintains the 3'-ends of eukaryotic chromosomes by adding tandem telomeric repeats. One striking features of telomerase that distinguish from the canonical RT is its capacity to precisely and repetitively replicate telomeric sequence without any additional bases. Previous studies have suggested that a duplex region of the RNA upstream of the template determine the template boundary. In this study, we characterized the polymerization properties of a reconstituted Candida telomerase containing a highly purified recombinant TERT and an in vitro transcribed telomerase RNA.Surprisingly, we found that the reconstituted telomerase displays several unanticipated properties. First, it functions as a reverse transcriptase by using just the template region of TER without the assistance of the non-template structural motifs of TER; Second, the DNA synthesis are halted at a defined template position, immediately before the rC²⁶¹-rC²⁶² dinucleotide pausing signal, and thirdly, a template stem can be unwound once the polymerization proceeds beyond the dinucleotide signal, a processes strictly depending on the presence of telomerase protein. Finally and even more surprisingly, Candida TERT was shown to mediate the extension of non-telomeric DNAs using non-TER RNA templates, thus essentially acting as a prototypical reverse transcriptase. These findings provide new insights on the molecular mechanisms of telomerase, and expand the repertoire of TERT-mediated reactions.To our surprised to find that Candida Pif1 had the exonuclease activity of 3'-5'in the study.Recent studies frequently found 3 '- 5' exonuclease activity in the polymerase and the helicase, in the study we reveal the pif1 family from bacteria to humans of highly conserved enzyme not only has the unwinding activity also has an exonuclease activity. However the exonuclease activity sequence not in the same region with helicase activity center and respective implementation of the different functions.After through mutation of two conserved metal binding position of acidic amino acid "E" into neutral amino acid "A" we found that exonuclease activity of Candida Pif1 basically disappeared. Before us more determined notonly Candida Pif1 has the function of helicase but also exonuclease. At the same time, we found out the Pif protein from 8 species according to the comparison of homologous sequences, and we found that bacteriassp?BsPif1?,Homo sapiens?hPif1?, Candida tropicalis?CtPif1? Saccharomyces cerevisiae?ScPif1??very weak?, which had no exonuclease activity. Meyerozyma guilliermondii?MgPif1?, Vanderwaltozyma polyspora?VpPif1?andCandida dubliniensis?CdPif1?. show the exonuclease activity. Pif1 with new activity enable us to reconsider what kind of role it plays in the cells, To remove the wrong base pairing that occurs in the telomerase polymerization, or to form a bTbble structure at telomere to protect telomere DNA to be removed. These new findings provide new insights into the role of DNA molecules in the study of telomerase aggregation and modification.