Analysis of Structure-Function Relationships in *Lambda and HK022 Site -Specific Recombination

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.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Microbiology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2015

Author dc:creator

• Swalla, Brian Melchior

Contributors dc:contributor

• Gardner, Jeffrey F.

• Biology, Microbiology

Language dc:language

eng

Identifier

(MiAaPQ)AAI3101977

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/86658