Date Available
12-14-2011
Year of Publication
2005
Document Type
Dissertation
College
Medicine
Department
Biochemistry
First Advisor
Guo-Min Li
Abstract
PCNA and RPA are required for DNA mismatch repair (MMR), but their rolesin the pathway are not fully understood. Using an affinity pull-down approach, weshow that (1) increased PCNA binding to DNA heteroduplexes is associated withthe appearance and accumulation of excision products; and (2) RPAphosphorylation occurs when DNA polymerase ?? binds to the DNA substrate. Wetherefore hypothesize that PCNA plays an important role in mismatch-provokedexcision and that RPA phosphorylation plays an important role in DNA resynthesis.To determine the role of PCNA in MMR, mismatch-provoked and nick-directedexcision was assayed in a cell-free system in the presence of the PCNA inhibitor,p21CIP1/WAF. We show that whereas PCNA is essential for 3' directed excision, it isdispensable for the 5' directed reaction, suggesting a differential role for PCNA inMMR. We further find that the PCNA-dependent pathway is the only pathway for3' directed excision, but there are at least two pathways for 5' directed excision,one of which is a PCNA-independent 5' excision pathway. To determine if RPAphosphorylation facilitates DNA resynthesis, a gap-filling assay was developedusing both a cell-free system and a purified system, and we demonstrate that RPAphosphorylation stimulates DNA polymerase ??-catalyzed resynthesis in bothsystems. Kinetic studies indicate that phosphorylated RPA has a lower affinity forDNA compared with un-phosphorylated RPA. Therefore, the stimulation ofresynthesis by phosphorylated RPA is likely due to the fact that phosphorylationpromotes the release of RPA from DNA, thereby making DNA template availablefor resynthesis.
Recommended Citation
Guo, Shuangli, "ROLE OF REPLICATION PROTEIN A (RPA) AND PROLIFERATING CELL NUCLEAR ANTIGEN (PCNA) IN DNA MISMATCH REPAIR" (2005). University of Kentucky Doctoral Dissertations. 258.
https://uknowledge.uky.edu/gradschool_diss/258
