Dr. David S. Pederson
DNA Replication and Repair in Chromatin
Current research in the Pederson lab focuses on two areas, control of DNA Replication and Base Excision Repair (BER) of damaged DNA in chromatin. Most of the DNA in eukaryotes is packaged by histone proteins into nucleosomes that, together with other (less abundant) proteins, make up chromatin. Although some factors appear able to bind DNA targets in nucleosomes, many cannot. Hence, processes such as transcription, DNA replication and repair generally entail the remodeling or disruption of nucleosomes.
Free radicals produced during the course of normal cellular metabolism are responsible for much of the roughly 20,000 oxidative lesions that occur each day in each human cell. Although we know that oxidative lesions are generally repaired in an error-free manner by BER enzymes, we know much less about how BER occurs in a chromatin millieu. Accordingly, our major goals are to identify rate-limiting steps in the BER of oxidative lesions in nucleosomes, and to identify factors that overcome these rate-limiting steps.
To investigate BER in chromatin, we assemble nucleosomes that contain defined oxidative lesions, at discrete positions within the nucleosome. By incubating these nucleosomes with highly purified human DNA repair enzymes, we have demonstrated that the complete, stepwise repair of lesions in nucleosomes can occur without irreversibly disrupting the nucleosome. Individual steps in the BER reaction are accompanied by a succession of enzyme-nucleosome ternary complexes that we have begun to characterize (Prasad et al., 2007; Odell et al., in preparation). We are also investigating dynamic behaviors of the nucleosome itself that facilitate access to lesions that would otherwise be sterically inaccessible (Prasad et al., manuscript under revision). Finally, to link results from our in vitro studies to the repair of oxidative lesions in nuclei, we have begun to tackle the question of how BER enzymes are able to efficiently locate lesions in a sea of undamaged DNA (Odell et al., 2010).
In the area of DNA replication, we are studying a protein, known as Clf1p (a yeast homolog of the crooked neck protein in Drosophila), that plays a critical role in assembly of pre-mRNA spliceosomes. We discovered that Clf1p also associates with origins of DNA replication, and is critical for efficient replication during S phase of the cell cycle (Zhu et al., 2002; Zhu et al., manuscript under revision). We are using molecular genetic techniques to test the hypothesis that Clf1p acts as a scaffold that helps recruit proteins that act in each of these two processes.
Odell ID, Newick K, Heintz NH, Wallace SS, Pederson DS Non-specific DNA binding interferes with the efficient excision of oxidative lesions from chromatin by the human DNA glycosylase, NEIL1. DNA Repair (Amst). 2010 Feb 4;9(2):134-43. Epub 2009 Dec 11. Erratum in: DNA Repair (Amst). 2010 Aug 5;9(8):938.
Prasad A, Wallace SS, Pederson DS. Initiation of base excision repair of oxidative lesions in nucleosomes by the human, bifunctional DNA glycosylase NTH1. Mol Cell Biol. 2007 Dec;27(24):8442-53
Zhu W, Rainville IR, Ding M, Bolus M, Heintz NH, Pederson DS. Evidence that the pre-mRNA splicing factor Clf1p plays a role in DNA replication in Saccharomyces cerevisiae. Genetics. 2002 Apr;160(4):1319-33.
Geraghty DS, Ding M, Heintz NH and Pederson DS. Premature structural changes at replication origins in a yeast minichromosome maintenance (MCM) mutant. J. Biol. Chem. 2000. Jun 16;275(24):18011-21.
Geraghty DS, Sucic H, Chen J, and Pederson DS. Evidence that partial unwrapping of DNA from nucleosomes facilitates the binding of heat shock factor following DNA replication in yeast. J. Biol. Chem., 1998, Aug 7;273(32):20463-20472.