This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Complete and accurate DNA replication once per cell cycle is essential to the maintenance of genetic integrity of any organism. Polymerase ?-Primase complex plays an important role in this process. It is involved in both the initiation of DNA replication at the origin and the subsequent elongation through synthesizing Okazaki fragment on the lagging strand of the replication fork. We are interested in studying the proteins that constitute this complex, their interaction and modifications through cell cycle, which may shed some light on the control of their functions. Using technologies previously developed in the lab, including genomic tagging in Saccharomyces cerevisiae, protein pull-out with rabbit IgG conjugated magnetic Dynabeads, and characterization of proteins and their posttranslational modification by mass spectrometry, we obtained biochemically active pol ? complex, confirmed its four-subunit composition, among which pol1- pol12 associate tightly while pri1- pri2 associate tightly, and observed the cell cycle-dependent phosphorylation of pol12 subunit, which potentially could be a mechanism to regulate Pol ? function through cell cycle. 7 potential phosphorylated peptides from pol12 digest were identified, indicating 7 possible phosphorylation sites. Current effort is focused on the identification of exact phosphorylation sites and their regulation through cell cycle. We are also experimenting with different methods for phosphorylation identification in general.
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