Project Details
Description
DESCRIPTION (adapted from applicant's abstract): The enzymatic synthesis of DNA
is a multistep process that requires sequential binding of substrate,
accompanied by several conformational changes within the enzyme protein. The
molecular mechanisms involved in these steps are not well understood. The
recent availability of a number of crystal structures of this class of enzymes,
however, has made a significant advancement whereby the basic molecular
mechanisms of DNA polymerization and their relationship to the structural
makeup of enzyme may be clarified at the atomic level. The major objective of
this proposal is to continue investigations on the biochemical, enzymological
and structural properties of the prototype enzyme, namely E. coli polymerase 1.
The choice of this enzyme in the proposed study is based on the fact that a)
the three dimensional anatomy of large fragment (Klenow enzyme) of pol I
family, complexed with substrates, has been resolved, b) significant
information regarding the process of substrate and template-primer binding by
pol I has been obtained from kinetic analysis, c) some of the sites (amino acid
residues) participating in the substrates and template binding have been
identified, d) a number of catalytic residues with some functional implication
have been identified by site-directed mutagenesis, and e) this enzyme serves as
the model system for mechanistic study of all DNA polymerases. In order to
identify and relate important structural domains that carry out specific
function in the catalysis of DNA synthesis, the following tripartite approach
will be used: i) site directed mutagenesis of amino acid residues in conserved
domains or implied by 3-D model structure examinations. An in depth analysis of
the properties of mutant enzyme will clarify the role for the desired amino
acid in specific domain structure, ii) photo-affinity labeling of enzyme
proteins with template-primers and identification of sites of enzyme and
template-primer contact and, iii) utilize all available structural information
concerning DNA polymerases in the interpretation of mutagenesis results as well
as to construct structural models which provide the detailed functional
participation of various domain structures in atomic details and permit
structural elucidation of the transition state, of the catalytic reaction. The
molecular mechanism and functional anatomy of DNA polymerase clarified in this
manner will lead to a better understanding of DNA replication, DNA repair and
mutagenic effects of chemicals and carcinogenesis.
Status | Finished |
---|---|
Effective start/end date | 1/1/90 → 8/31/06 |
Funding
- National Institute of General Medical Sciences: $274,750.00
- National Institute of General Medical Sciences: $274,750.00
- National Institute of General Medical Sciences: $274,750.00
- National Institute of General Medical Sciences: $274,750.00
ASJC
- Genetics
- Molecular Biology
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