Objectives and Methods
We will determine how the DNA repair enzymes
of the apurinic/apyrimidinic endonuclease (APE) family recognize the structure
and dynamics of DNA damage sites resulting from low dose ionizing radiation
(IR). Accurate crystal structures exist for the bacterial member of this
family (Mol
et al., 1995) and the human APE1 in complex with a synthetic, cleavable
tetrahydrofuran analogue of an AP site (Mol
et al., 2000). These crystal structures are used as the basis for homology
modeling of other proteins of this family. We are running molecular dynamic
simulations of wild type and mutant APE1 to analyze the flexibility of
catalytically important residues and to predict alternative conformations
of these proteins which might be important in recognition of damaged DNA
sites. Blast and Psi-blast searching with the human APE1 sequence has revealed
many related protein sequences, which are being used to locate structurally
and functionally important residues. We further developing our expert system,
MASIA (Hänggi
and Braun, 1994; Zhu et al., 1999b), to identify motifs of conserved amino
acids. These are used to identify other relatives of the APE family in
the human genome family. The predictions we make will be tested in a unique
complementation assay in APE deficient bacterial cells. We pursue the following
specific aims:
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To use molecular dynamics simulations of wild type
human APE1/DNA complex and of mutants thereof to define energetically accessible
conformations near the known X-ray structure of the complexes
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To generate 3D models of APE1 mutants to dissect the functional
and structural role of amino acids near the active site. Predictions made
from these computations will be tested by site directed mutagenesis.
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To use our MASIA
program to automatically identify functional motifs by searching aligned
sequences of the known AP-endonucleases and lyases and to test these predictions
with SDM and APE functional assays
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To search the human genome for related proteins containing
these motifs.
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To develop a vector based representation of the APE1 family
that accounts for the states of amino acids contributing to its function
and structure, and to use this representation to account for the different
activities of the mammalian and bacterial enzymes.
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To model the 3D structure of the N-terminal 6 kD region of
human APE and search for potential protein binding partners.