Molecular Determinants of DNA Sliding and Hopping by a DNA Repair Glycosylase

Summary

Principal Investigator: MENG MENG ROWLAND
Abstract: DESCRIPTION (provided by applicant): Aberrant uracils in human genomic DNA lead to G:C to A:T transversion mutation, the most frequent mutation found in many cancers. Human uracil DNA glycosylase (hUNG) searches and removes uracil bases from deoxyribose phosphate backbone using a remarkable search mechanism that allows the enzyme to rapidly locate rare lesion sites in a background of roughly 6 billion normal base pairs in the human genome. This general search mechanism has been referred to as "facilitated diffusion", where proteins utilize the DNA chain as a track to accelerate location of their specific sites. Facilitated diffusion uses two microscopic transfer pathways: one-dimensional "sliding" and three-dimensional "hopping". Although numerous studies have established the existence of such a general mechanism, the details of "facilitated diffusion" remain mysterious. The focus of this proposal is to elucidate th molecular interactions that are important for DNA chain tracking by hUNG. Our recently developed "molecular clock" (MC) approach utilizes a small molecule trap to capture transient hopping enzymes while having minimal effect on the sliding ones, thus allowing the dissection of the two tracking pathways individually. Key parameters that can be probed using this approach include the calculation of mean sliding distance, the average distance an enzyme hops away from DNA, and the 1D diffusion constant for DNA sliding. In the first aim, we will use MC approach to test several key aspects of DNA tracking. First, to test the role of DNA phosphate electrostatics, we will insert neutral methylphosphonate linkages in the DNA backbone between two uracil target sites separated by a set number of base pairs, and then measure the effect of charge ablation on the probability that hUNG will slide between the sites. Second, we will test a novel hypothesis that directionally-biased transfer can occur (even in the absence of an energy- providing cofactor) if thermodynamically stable binding sites are inserted between the two uracil target sites. This aim will involve the insertion of spaced high-affinity tetrahydrofuran abasic sie analogues between the uracil sites. The prediction is that a biased walk will occur via high-affinity "island transfer". A biased-walk using basic sites is highly relevant to the in vivo situaion where hUNG must locate and excise clustered uracil sites such as in the process of Ig somatic hypermutation. The second aim is to engineer hUNG-peptide tail variants to have enhanced tracking abilities. Fusion proteins between hUNG and short, positively charged peptides will be constructed using expressed protein ligation technology (EPL) and/or chemical ligation. These peptides will be derived from several DNA binding proteins (p53, HOXD9, H3), and are chosen because they represent various sizes and overall charge, and are known to affect DNA binding and association kinetics. We will measure the fundamental tracking parameters with these variants, which will directly test how the residence time on nonspecific DNA, and the 1D diffusion constant for sliding affect the efficiency of damage repair both in vitro and in vivo.
Funding Period: 2013-01-01 - 2014-12-31
more information: NIH RePORT

Research Grants

Detail Information

Research Grants30

  1. Structure and Mechanism in DNA Excision Repair
    GREGORY LAWRENCE VERDINE; Fiscal Year: 2013
    ..Together, these studies aim to provide a comprehensive molecular-level framework for understanding two very important but very different strategies for seeking out and destroying genotoxic lesions in DNA. ..
  2. Somatic hypermutation and class switching in autoimmunity
    Paolo Casali; Fiscal Year: 2013
    ....
  3. Coupling of structure and dynamics in RNA catalysis
    BARBARA LYNN GOLDEN; Fiscal Year: 2013
    ..We anticipate that the results of this study will provide clues as to the catalytic strategies of many ribozymes, some of which will be therapeutic targets. ..
  4. Elucidating the Chemistry and Biology of Nucleic Acid Cytidine Deaminases in HIV
    RAHUL MANU KOHLI; Fiscal Year: 2013
    ..abstract_text> ..
  5. MOLECULAR GENETICS OF HSV DNA POLYMERASE GENE
    James M Hogle; Fiscal Year: 2013
    ..The research proposed should not only provide information that could aid in drug discovery and understanding how viruses become resistant to current drugs, but aims directly to discover new anti-herpesvirus drugs. ..
  6. Resolution Mechanisms in Acute Inflammation: Resolution Pharmacology
    CHARLES NICHOLAS SERHAN; Fiscal Year: 2013
    ..Selected synthetic SPM will be scaled-up for demonstration of their unique mode of action in vivo in a resolution pharmacology core using experimental disease models. Our broad goal is to bring forth new treatments in resolution. ..
  7. Spatial and Temporal Regulation of Angiogenesis
    HAROLD FISHER DVORAK; Fiscal Year: 2013
    ..abstract_text> ..