The Exploration On The Mechanism Of Ubiquitination Of Cdc45 In DNA Replication

DNA replication refers to the process of synthesizing two progeny DNA strands by using a parental DNA molecule as a template during the cell division.In eukaryotic cells,the DNA helicase complex(Cdc45–Mcm–GINS helicase complex,referred to as CMG)is the major complex which forms and maintains the replication fork during DNA replication.Cdc45 is one of the important components of CMG and plays an important role in regulating DNA replication.Ubiquitination refers to the process in which ubiquitin molecules are covalently bound to target proteins under the action of a series of enzymes,and participates in the regulation of almost all life activities of eukaryotes.Many studies have found that in the process of DNA replication and DNA damage repair,a variety of related proteins are regulated by ubiquitination modification to ensure the stability of the genome.In this study,we used Saccharomyces cerevisiae as a research object to investigate whether Cdc45 has ubiquitination during DNA replication,and to study its regulatory mechanism and its role in DNA replication.1)First,we identified the modified band of Cdc45 as a ubiquitinated band by the exogenously induced over-expression,and screened the ubiquitination sites K156 and K643 on Cdc45 via point mutation;subsequently,we utilized the cell cycle synchronization and gene knockout,found that Cdc45 was ubiquitinated by the ubiquitin ligase Dia2 during DNA replication,and 2)then the ubiquitinated Cdc45 was deubiquitinated by the deubiquitinating enzyme Doa4.3)Finally,by flow cytometry analysis of DNA synthesis,we found that inhibition of ubiquitination of Cdc45 leads to a prolonged DNA replication cycle,but knockdown of ubiquitinating enzymes can compensate for this deficiency.This study found that Cdc45 is regulated by ubiquitination and deubiquitination during DNA replication,and it is preliminarily confirmed that this ubiquitination regulation mode is involved in the regulation of DNA replication,providing a new basis for re-recognizing the role of Cdc45 in DNA replication.