If DNA could adopt only the structure of DNA with which we are most familiar - the canonical B-form double helix - it would be nearly devoid of biologic function. The ability of proteins to coerce DNA into a variety of non-canonical structures is an absolute prerequisite for many of the most important processing events that take place on the genome, including replication, transcription initiation, DNA repair, recombination, supercoiling, and packaging into chromatin. Although most of these processes are well-known at a descriptive level, much less is known about the pathways leading to DNA deformation and the associated energetic costs. The long-term goals of this research are to understand how the structure of DNA responds to torsional stress, and to elucidate the mechanisms by which proteins bring about alterations of DNA structure. The proposed studies will address this issue by trapping proteins in the act of inducing DNA distortion. A new and powerful technology that relies on the thermodynamically-driven formation of a disulfide crosslink between proteins and DNA will be employed to freeze otherwise transient complexes, thereby enabling high-resolution structural studies. The specific systems chosen for study are: RAG recombinase. This enzyme catalyzes a spectacular rearrangement of backbone connectivity in DNA, which is essential for the generation of immunologic diversity. Chemical and structural methods will be used to study the recombinase reaction mechanism in detail. Uracil DNA glycosylase. This enzyme initiates the repair of mutagenic uracil residues in DNA. How the enzyme locates these residues amidst the vast excess of normal DNA is a subject of the proposed investigation. HMGI-(Y). This protein stimulates the biological and biochemical activities of numerous diverse transcription factors and DNA- modifying enzymes. The mechanisms of stimulation is believed to involve manipulation of DNA architecture. The proposed experiments aim to resolve key issues regarding the DNA-binding mode of HMG-I(Y) and the mechanism by which it stimulates transactivation of the interferon-beta promoter by NF-kappaB.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM044853-09
Application #
6022019
Study Section
Special Emphasis Panel (ZRG1-MCHA (01))
Project Start
1990-08-31
Project End
2003-07-31
Budget Start
1999-08-01
Budget End
2000-07-31
Support Year
9
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Harvard University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
071723621
City
Cambridge
State
MA
Country
United States
Zip Code
02138
Sung, Rou-Jia; Zhang, Michael; Qi, Yan et al. (2013) Structural and biochemical analysis of DNA helix invasion by the bacterial 8-oxoguanine DNA glycosylase MutM. J Biol Chem 288:10012-23
Didovyk, Andriy; Verdine, Gregory L (2012) Structural origins of DNA target selection and nucleobase extrusion by a DNA cytosine methyltransferase. J Biol Chem 287:40099-105
Qi, Yan; Nam, Kwangho; Spong, Marie C et al. (2012) Strandwise translocation of a DNA glycosylase on undamaged DNA. Proc Natl Acad Sci U S A 109:1086-91
Crenshaw, Charisse M; Nam, Kwangho; Oo, Kimberly et al. (2012) Enforced presentation of an extrahelical guanine to the lesion recognition pocket of human 8-oxoguanine glycosylase, hOGG1. J Biol Chem 287:24916-28
Gude, Lourdes; Berkovitch, Shaunna S; Santos, Webster L et al. (2012) Mapping targetable sites on human telomerase RNA pseudoknot/template domain using 2'-OMe RNA-interacting polynucleotide (RIPtide) microarrays. J Biol Chem 287:18843-53
Sung, Rou-Jia; Zhang, Michael; Qi, Yan et al. (2012) Sequence-dependent structural variation in DNA undergoing intrahelical inspection by the DNA glycosylase MutM. J Biol Chem 287:18044-54
Qi, Yan; Spong, Marie C; Nam, Kwangho et al. (2010) Entrapment and structure of an extrahelical guanine attempting to enter the active site of a bacterial DNA glycosylase, MutM. J Biol Chem 285:1468-78
Bowman, Brian R; Lee, Seongmin; Wang, Shuyu et al. (2010) Structure of Escherichia coli AlkA in complex with undamaged DNA. J Biol Chem 285:35783-91
Qi, Yan; Spong, Marie C; Nam, Kwangho et al. (2009) Encounter and extrusion of an intrahelical lesion by a DNA repair enzyme. Nature 462:762-6
Blainey, Paul C; Luo, Guobin; Kou, S C et al. (2009) Nonspecifically bound proteins spin while diffusing along DNA. Nat Struct Mol Biol 16:1224-9

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