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https://hdl.handle.net/2142/86699
Description
Title
Characterization of IntDOT, the CTnDOT Integrase
Author(s)
Malanowska, Karolina Ann
Issue Date
2007
Doctoral Committee Chair(s)
Abigail Salyers
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
Sequence analysis revealed that the integrase of the Bacteroides conjugative transposon CTnDOT (IntDOT) might be a member of the tyrosine recombinase family because IntDOT has five of six highly conserved residues found in the catalytic domains of tyrosine recombinases. Yet, IntDOT catalyzes a reaction that appears to differ in some respects from well-studied tyrosine recombinases such as that of phage lambda. To assess the importance of the conserved residues, I changed residues in IntDOT that align with conserved residues in the tyrosine recombinases. Some substitutions resulted in a complete loss or significant decrease of integration activity in vivo. The ability of the mutant proteins to cleave and ligate CTnDOT attachment site (attDOT) DNA in vitro in general paralleled the in vivo results. I also showed that the predicted three dimensional structure of the IntDOT core binding domain is similar to that of well-studied tyrosine recombinases. My results confirmed the hypothesis that IntDOT belongs to the tyrosine recombinase family. Previous DNA sequence analyses showed that CTnDOT att sites contain 5 base pair non-homologous coupling sequences which were assumed to define the putative staggered sites of cleavage. However, my cleavage assays showed that one of the cleavage sites is two base pairs away from the junction of CTnDOT and coupling sequence DNA. This site is in a region of homology that is conserved in CTnDOT att sites. I tested the ability of IntDOT to cleave and ligate activated attDOT substrates in the presence of mismatches. Unlike other tyrosine recombinases, the results revealed that IntDOT is able to perform ligation reactions even when all the bases within the crossover region are mispaired. I also showed that there is a strong bias in the order of strand exchanges during integrative recombination. The top strands are exchanged first in reactions that require two base pairs of homology between the partner sites adjacent to the sites of cleavage. The bottom strands are exchanged next in reactions that do not require homology between the partner sites. This mode of coordination of the strand exchanges in unique among tyrosine recombinases.
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