Structure and Mechanism in DNA Excision Repair

Summary

Principal Investigator: GREGORY LAWRENCE VERDINE
Abstract: Spontaneous damage to the four bases of DNA is a major cause of the mutations that give rise to cancer. Most of these genetic lesions are corrected by either of two pathways, base-excision DNA repair (BER) or nucleotide-excision DNA repair (NER). BER is primarily responsible for the repair of nucleobases having relatively small, simple changes with respect to the normal nucleobases in DNA, whereas NER repairs a wide variety of bulky nucleobase lesions such as thymine dimers. The key components of BER are DNA glycosylases, professional lesion-hunting enzymes that scan the genome in search of particular kinds of base damage, then catalyze excision of the damaged base from the DNA backbone. NER does not have such specialized lesion-recognition enzymes, but instead employs two proteins, UvrA and UvrB, to search cooperatively for damaged nucleobases of many different kinds, the only common feature being that they are bulky. The long-term goals of our studies are to understand how these enzymes locate their particular kinds of damage amidst the vast expanse of normal DNA, and how they catalyze repair of the damage once having located it. A comprehensive, fundamental understanding of DNA damage recognition and removal represents the solution to a major aspect of the tumorigenesis puzzle. In the proposed studies, we will study base-excision repair proteins of the so-called GO system that are responsible for either direct repair of the highly mutagenic lesion 8-oxoguanine (oxoG) - the hOgg1 enzyme in eukaryotes and MutM in prokaryotes - or repair of the mutagenic adenine in oxoG:A base-pair resulting from mis-replication of unrepaired oxoG lesions, catalyzed by the MutY protein (hMYH in humans). We also propose to investigate lesion recognition by glycosylases that repair a variety of genotoxic methylated adducts in DNA (the AlkA protein in bacteria and Aag in humans). On the NER front, we will study the early events in the pathway leading to the loading of UvrB onto a lesion and recruitment of a UvrB-dependent endonuclease, UvrC, which cleaves the DNA backbone at sites flanking the lesion. Here we outline a broad-based, interdisciplinary approach that employs the use of chemical crosslinking and synthetic, photoactive nucleobase analogs to trap intermediates in the BER and NER repair pathways. To understand dynamic aspects of lesion recognition, we will employ time-resolved X-ray crystallography and single-molecule DNA tracking studies. 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.
Funding Period: 2003-04-01 - 2013-11-30
more information: NIH RePORT

Top Publications

  1. pmc A base-excision DNA-repair protein finds intrahelical lesion bases by fast sliding in contact with DNA
    Paul C Blainey
    Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA
    Proc Natl Acad Sci U S A 103:5752-7. 2006
  2. ncbi Small-angle X-ray scattering reveals architecture and A₂B₂ stoichiometry of the UvrA-UvrB DNA damage sensor
    Danaya Pakotiprapha
    Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachussets 02138, USA
    Proteins 81:132-9. 2013
  3. pmc Enforced presentation of an extrahelical guanine to the lesion recognition pocket of human 8-oxoguanine glycosylase, hOGG1
    Charisse M Crenshaw
    Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA
    J Biol Chem 287:24916-28. 2012
  4. pmc Sequence-dependent structural variation in DNA undergoing intrahelical inspection by the DNA glycosylase MutM
    Rou Jia Sung
    Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA
    J Biol Chem 287:18044-54. 2012
  5. pmc Encounter and extrusion of an intrahelical lesion by a DNA repair enzyme
    Yan Qi
    Graduate Program in Biophysics, Harvard Medical School, Boston, Massachusetts 02115, USA
    Nature 462:762-6. 2009
  6. pmc Analysis of an anomalous mutant of MutM DNA glycosylase leads to new insights into the catalytic mechanism
    Kwangho Nam
    Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA
    J Am Chem Soc 131:18208-9. 2009
  7. pmc Nonspecifically bound proteins spin while diffusing along DNA
    Paul C Blainey
    Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA
    Nat Struct Mol Biol 16:1224-9. 2009
  8. pmc Entrapment and structure of an extrahelical guanine attempting to enter the active site of a bacterial DNA glycosylase, MutM
    Yan Qi
    Graduate Program in Biophysics, Harvard Medical School, Boston, Massachusetts 02115, USA
    J Biol Chem 285:1468-78. 2010
  9. pmc Atomic substitution reveals the structural basis for substrate adenine recognition and removal by adenine DNA glycosylase
    Seongmin Lee
    Departments of Stem Cell and Regenerative Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA
    Proc Natl Acad Sci U S A 106:18497-502. 2009
  10. pmc A structural model for the damage-sensing complex in bacterial nucleotide excision repair
    Danaya Pakotiprapha
    Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA
    J Biol Chem 284:12837-44. 2009

Detail Information

Publications16

  1. pmc A base-excision DNA-repair protein finds intrahelical lesion bases by fast sliding in contact with DNA
    Paul C Blainey
    Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA
    Proc Natl Acad Sci U S A 103:5752-7. 2006
    ..5 kcal/mol (1 kcal = 4.2 kJ). This nearly barrierless Brownian sliding indicates that DNA glycosylases locate lesion bases by a massively redundant search in which the enzyme selectively binds 8-oxoguanine under kinetic control...
  2. ncbi Small-angle X-ray scattering reveals architecture and A₂B₂ stoichiometry of the UvrA-UvrB DNA damage sensor
    Danaya Pakotiprapha
    Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachussets 02138, USA
    Proteins 81:132-9. 2013
    ..The current SAXS analysis failed to detect significant changes to the structure as a function of nucleotide...
  3. pmc Enforced presentation of an extrahelical guanine to the lesion recognition pocket of human 8-oxoguanine glycosylase, hOGG1
    Charisse M Crenshaw
    Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA
    J Biol Chem 287:24916-28. 2012
    ..This study demonstrates that even if hOGG1 mistakenly inserts a normal base into its active site, the enzyme can still reject it on the basis of catalytic incompatibility...
  4. pmc Sequence-dependent structural variation in DNA undergoing intrahelical inspection by the DNA glycosylase MutM
    Rou Jia Sung
    Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA
    J Biol Chem 287:18044-54. 2012
    ....
  5. pmc Encounter and extrusion of an intrahelical lesion by a DNA repair enzyme
    Yan Qi
    Graduate Program in Biophysics, Harvard Medical School, Boston, Massachusetts 02115, USA
    Nature 462:762-6. 2009
    ..All-atom computer simulations show the pathway by which encounter of the enzyme with the lesion causes extrusion from the DNA duplex, and they elucidate the critical free energy difference between oxoG and G along the extrusion pathway...
  6. pmc Analysis of an anomalous mutant of MutM DNA glycosylase leads to new insights into the catalytic mechanism
    Kwangho Nam
    Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA
    J Am Chem Soc 131:18208-9. 2009
    ....
  7. pmc Nonspecifically bound proteins spin while diffusing along DNA
    Paul C Blainey
    Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA
    Nat Struct Mol Biol 16:1224-9. 2009
    ..The average free-energy barrier for sliding along the DNA was 1.1 +/- 0.2 k(B)T. Such small barriers facilitate rapid search for binding sites...
  8. pmc Entrapment and structure of an extrahelical guanine attempting to enter the active site of a bacterial DNA glycosylase, MutM
    Yan Qi
    Graduate Program in Biophysics, Harvard Medical School, Boston, Massachusetts 02115, USA
    J Biol Chem 285:1468-78. 2010
    ..The structure of this MutM-extrahelical G complex provides insights into the mechanism MutM employs to discriminate against extrahelical normal DNA bases and into the base extrusion process in general...
  9. pmc Atomic substitution reveals the structural basis for substrate adenine recognition and removal by adenine DNA glycosylase
    Seongmin Lee
    Departments of Stem Cell and Regenerative Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA
    Proc Natl Acad Sci U S A 106:18497-502. 2009
    ....
  10. pmc A structural model for the damage-sensing complex in bacterial nucleotide excision repair
    Danaya Pakotiprapha
    Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA
    J Biol Chem 284:12837-44. 2009
    ..The crystal structure and accompanying biochemical analyses suggest a model for the complete damage-sensing complex...
  11. pmc Synthesis and structure of duplex DNA containing the genotoxic nucleobase lesion N7-methylguanine
    Seongmin Lee
    Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA
    J Am Chem Soc 130:11570-1. 2008
    ..These observations suggest that AlkA and Aag must perform a structurally invasive interrogation of DNA in order to detect the presence of intrahelical m7dG lesions...
  12. pmc Structure of the E. coli DNA glycosylase AlkA bound to the ends of duplex DNA: a system for the structure determination of lesion-containing DNA
    Brian R Bowman
    Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA
    Structure 16:1166-74. 2008
    ..The structures of 8-oxoguanine provide a correct atomic-level view of this important endogenous lesion in DNA...
  13. pmc Trapping and structural elucidation of a very advanced intermediate in the lesion-extrusion pathway of hOGG1
    Seongmin Lee
    Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA
    J Am Chem Soc 130:7784-5. 2008
    ....
  14. pmc Crystal structure of Bacillus stearothermophilus UvrA provides insight into ATP-modulated dimerization, UvrB interaction, and DNA binding
    Danaya Pakotiprapha
    Department of Molecular and Cellular Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA
    Mol Cell 29:122-33. 2008
    ..Structural analysis, biochemical experiments, surface electrostatics, and sequence conservation form the basis for models of ATP-modulated dimerization, UvrA-UvrB interaction, and DNA binding during the search for lesions...
  15. ncbi Structural characterization of human 8-oxoguanine DNA glycosylase variants bearing active site mutations
    Christopher T Radom
    Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138, USA
    J Biol Chem 282:9182-94. 2007
    ..The latter structure offers a view of the latest stage in the base extrusion pathway yet observed, and its lack of catalytic activity demonstrates that the transition state for displacement of the lesion base is geometrically demanding...
  16. pmc Structural and biochemical analysis of DNA helix invasion by the bacterial 8-oxoguanine DNA glycosylase MutM
    Rou Jia Sung
    Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA
    J Biol Chem 288:10012-23. 2013
    ....