Efficient coordination of base excision repair (BER) in vivo involves a complex mosaic of pathways. Project 2 of the SBDR Program studies interactions governing pathway choice in BER and coordinating it with other DNA repair pathways and other vital DNA interactions. This research is based on the tenets that (1) BER must be a highly coordinated process to prevent toxic release of unchaperoned repair intermediates; (2) BER must repair lesions that block RNA polymerases in a transcription- coupled repair (TCR) process; and (3) BER should be coordinated with DNA replication to preferentially repair nascent strands in a replication- associated repair (RAR) process. These interactions are mediated through protein/protein and/or protein/DNA interfaces and may involve either transient hand-offs of DNA repair intermediates, recruitment of proteins to lesions, or stable retention of BER enzymes in DNA polymerse complexes in the presence of DNA, identify conditions for stable complexes, and determine the structural basis for the interactions. The success of this project relies heavily on the EMB core for determining conditions for expressing proteins and protein fragments that assemble into complexes and on the SCB core for structural analyses.
Each aim i s directed at understanding the modulation of BER enzymes at different stages in the pathway.
Aim 1 focuses on the stimulation in activity of glycosylases involved in oxidative repair by XPG, a protein required for TCR, and by APE1, the next protein in the BER pathway.
Aim 2 examines EXP, FEN1, and PCNA interactions with APE1. FEN1 and PCNA follow APE1 in the BER pathway.
Aim 3 investigates the mechanism of long patch BER by defining interactions between APE1, FEN1, PCNA, and DNA Poldelta.
Aim 4 is directed at understanding the essential role of MutS proteins in TC-BER by analyzing the observed stimulation of MutS binding to DNA by XPG and NTH.
Aim 5 tests the RAR hypothesis that BER glycosylases preferentially repair nascent strands an that this preference is encoded in glycosylases preferentially repair nascent strands and that this preference is encoded in glycosylase interactions with certain components of the DNA replication machinery, RPA, PCNA, and Pol delta. Through quantitative characterization of dynamic complex assemblies coupled with high (X-ray crystallography) and low (EM) resolution structural determinations, results from these studies will go beyond enzymatic steps for BER to the complex mosaic of multiple DNA repair pathways. Our studies are strongly complementary to those of BER in Project 1 and of mismatch repair in Project 5. Since there is growing evidence for interplay between TCR and double-strand break repair pathways, our studies of XPG are also relevant to Projects 3 and 4.
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