Incorrect enzymatic transformation of DNA, may lead to DNA damage, somatic mutation, cancer, genetic disease, aging, and death. Numerous nucleases involved in DNA repair, restriction, and recombination (""""""""RRR"""""""") have been widely used to carry out genetic manipulations in vivo and in vitro, to perform medical diagnostics, and as model systems to study enzymatic reactions. This large class of enzymes is therefore of central importance in medicine and biotechnology. Several RRR nucleases were found to exhibit different 3D folds, typically of the alpha-beta class. Our understanding of sequence-structure-function relationships in these enzymes is severely limited by the slow progress of the structure determination - for most of them the 3D structure remains unknown. This project has two goals: 1) to develop a computational method that generates an experimentally validated model of the protein 3D structure, and 2) to apply this method to determine protein folds of RRR nucleases. For members of all RRR nuclease families, large ensembles of models will be generated using protein fold-recognition methods and the de novo structure prediction algorithm ROSETTA. At least 10 candidates for different 3D folds will be selected and probed by cross-linking, chemical modification and mutagenesis. Best models will be identified based on their fit with the experimental data and further tested by additional experiments. The results will advance our understanding of the structural diversity of RRR nucleases and will provide a structural platform for further studies of the processes of DNA repair, restriction, and recombination. Likely candidates for novel folds will be identified, which could be targeted for structure determination by X-ray crystallography. The computer software and the research protocol developed during this study will yield a predictive method for protein structure modeling that will be broadly applicable to all proteins.

Agency
National Institute of Health (NIH)
Institute
Fogarty International Center (FIC)
Type
Small Research Grants (R03)
Project #
5R03TW007163-03
Application #
7209825
Study Section
International and Cooperative Projects 1 Study Section (ICP)
Program Officer
Katz, Flora N
Project Start
2004-12-15
Project End
2007-11-30
Budget Start
2006-12-01
Budget End
2007-11-30
Support Year
3
Fiscal Year
2007
Total Cost
$31,575
Indirect Cost
Name
University of Washington
Department
Biochemistry
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Obarska-Kosinska, Agnieszka; Taylor, James E; Callow, Philip et al. (2008) HsdR subunit of the type I restriction-modification enzyme EcoR124I: biophysical characterisation and structural modelling. J Mol Biol 376:438-52
Fukuda, Eri; Kaminska, Katarzyna H; Bujnicki, Janusz M et al. (2008) Cell death upon epigenetic genome methylation: a novel function of methyl-specific deoxyribonucleases. Genome Biol 9:R163
Orlowski, Jerzy; Bujnicki, Janusz M (2008) Structural and evolutionary classification of Type II restriction enzymes based on theoretical and experimental analyses. Nucleic Acids Res 36:3552-69
Vasu, Kommireddy; Saravanan, Matheshwaran; Bujnicki, Janusz M et al. (2008) Structural integrity of the beta beta alpha-Metal finger motif is required for DNA binding and stable protein-DNA complex formation in R.KpnI. Biochim Biophys Acta 1784:269-75
Orlowski, Jerzy; Boniecki, Michal; Bujnicki, Janusz M (2007) I-Ssp6803I: the first homing endonuclease from the PD-(D/E)XK superfamily exhibits an unusual mode of DNA recognition. Bioinformatics 23:527-30
Jakubauskas, Arturas; Giedriene, Jolanta; Bujnicki, Janusz M et al. (2007) Identification of a single HNH active site in type IIS restriction endonuclease Eco31I. J Mol Biol 370:157-69
Kosinski, Jan; Kubareva, Elena; Bujnicki, Janusz M (2007) A model of restriction endonuclease MvaI in complex with DNA: a template for interpretation of experimental data and a guide for specificity engineering. Proteins 68:324-36
Ibryashkina, Elena M; Zakharova, Marina V; Baskunov, Vladimir B et al. (2007) Type II restriction endonuclease R.Eco29kI is a member of the GIY-YIG nuclease superfamily. BMC Struct Biol 7:48
Schafer, Patrick; Cymerman, Iwona A; Bujnicki, Janusz M et al. (2007) Human lysosomal DNase IIalpha contains two requisite PLD-signature (HxK) motifs: evidence for a pseudodimeric structure of the active enzyme species. Protein Sci 16:82-91
Pietal, Michal J; Tuszynska, Irina; Bujnicki, Janusz M (2007) PROTMAP2D: visualization, comparison and analysis of 2D maps of protein structure. Bioinformatics 23:1429-30

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