DNA topoisomerases, recombinases, endonucleases and ligases are basic to the genetic machinery of the cell. These enzymes are ubiquitous and essential for replication, transcription, recombination, and repair of DNA. Therefore, understanding their mechanisms of action is important for understanding the basis for genetic instability diseases such as cancer. Endonuclease NaeI appears to hold the key that relates these DNA processing enzymes to each other. No sequence similarity is detectable between NaeI and the topoisomerases and recombinases. A stretch of 10 amino acids in NaeI, however, has significant similarity to the adenylation/joining active site of DNA ligase I. A single L43K amino acid change within this region of NaeI radically transforms NaeI activity from endonuclease to that of a DNA topoisomerase and recombinase. The biochemical basis for this altered activity is unknown and the mechanism of this novel topoisomerase remains to be elucidated. The discovery of topoisomerase and recombinase activity within NaeI endonuclease unites what were previously believed to be independently derived DNA processing activities.
The specific aims of this proposal are to: (i) characterize the covalent attachment between NaeI/NaeI-43K and DNA; (ii) biochemically map the domain structures of NaeI and NaeI-L43K by limited proteolysis; (iii) solve the three-dimensional structure of NaeI protein complexed with a DNA fragment; (iv) improve the expression of NaeI-43K in vitro in order to isolate large amounts of this protein for crystallization studies, and (v) explore the structure-function relationships, implied by the crystal structures, by mutagenic and biochemical means.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM052123-05
Application #
2758465
Study Section
Molecular Biology Study Section (MBY)
Project Start
1995-01-01
Project End
2002-12-31
Budget Start
1999-01-01
Budget End
1999-12-31
Support Year
5
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
078861598
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599