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.
| Identifer | oai:union.ndltd.org:uky.edu/oai:uknowledge.uky.edu:gradschool_diss-1261 |
| Date | 01 January 2005 |
| Creators | Guo, Shuangli |
| Publisher | UKnowledge |
| Source Sets | University of Kentucky |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | University of Kentucky Doctoral Dissertations |
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