Analysis of Structure-Function Relationships in *Lambda and HK022 Site -Specific Recombination
Swalla, Brian Melchior
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https://hdl.handle.net/2142/86658
Description
Title
Analysis of Structure-Function Relationships in *Lambda and HK022 Site -Specific Recombination
Author(s)
Swalla, Brian Melchior
Issue Date
2003
Doctoral Committee Chair(s)
Gardner, Jeffrey F.
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Microbiology
Language
eng
Abstract
This thesis presents a multifaceted analysis of protein structure-function relationships pertaining to the Lambda and HK022 Integrase proteins (Int). First, homology-based modeling was used to create a three-dimensional model of the Lambda Int core-binding (CB) domain. The model suggests that all prokaryotic Tyrosine recombinases share a common CB domain fold, and facilitates interpretation of pre-existing genetic and biochemical data by providing a structural framework within which mutant phenotypes may be investigated. Second, Lambda and HK022 Int core-type DNA binding specificity was examined by employing challenge phages to isolate gain-of-function HK022 Int mutants that recognize the Lambda B' core site. The HK-Int D99 residue was identified as a negative specificity determinant that apparently contacts the bound core-type DNA site directly. Third, an exceptional mutation in Lambda Int, E47K, that displayed an excision-specific recombination defect was characterized by comparing the effects of various amino-acid substitutions at this position. A negatively charged residue was required for both cooperative Int-Xis and Int-Int binding, suggesting that E47 participates in two alternate types of cooperative binding contacts depending upon the binding context. Fourth, previously isolated Lambda Int mutants were characterized that resolved Holliday junctions with biases different from the wild-type Int. The T168N and E153K substitutions may directly affect cooperative interactions between Int proteins bound to the Holliday junction intermediate. The V175E substitution appears to change the structural relationship between the Int CB and catalytic domains by affecting the structure of the linker region that connects them. Collectively, these studies have yielded detailed information about the structural and functional nature of the Lambda and HK022 Ints that will be important for future studies investigating the regulation and dynamics of the higher-order intasome complexes in which these enzymes function.
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