A study of DNA-DNA interactions during bacteriophage lambda integrative recombination
Sun, Ke-Qin
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Permalink
https://hdl.handle.net/2142/23303
Description
Title
A study of DNA-DNA interactions during bacteriophage lambda integrative recombination
Author(s)
Sun, Ke-Qin
Issue Date
1990
Doctoral Committee Chair(s)
Gumport, Richard I.
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Language
eng
Abstract
An in vitro integrative recombination system was used to study DNA-DNA interactions during site-specific recombination in bacteriophage lambda. Oligodeoxynucleotides 28 bases long containing either the top strand or the bottom strand sequences of the bacterial attachment site (attB) with or without base substitutions in the core region were chemically synthesized. Annealed oligodeoxynucleotide attB sites containing sequences with perfect base pairs or one mismatched base apposition were shown to be proficient in integrative recombination. Supercoiled circular DNA molecules 713 base pairs long containing bacteriophage lambda attachment (attP) sites were constructed by cloning, annealing of the single strands, restriction digestion, and intramolecular ligation. These attP sites, with or without a mismatched base pair in the core region, are also proficient in integrative recombination. Recombination between perfectly base paired attB and supercoiled attP containing mismatched base pairs in the core region was less efficient than that between the wild-type att sites indicating that mismatches in the core region of attP are disrupting normal interactions. Recombination with attP and attB substrates, both containing mismatched base pairs in the overlap region, demonstrated that sequence homology at positions -2 or 0 is not required for the first strand exchange during integrative recombination. The sequence homology at those positions is, however, required for branch migration of the Holliday-structure intermediate formed by the first strand exchange. The results also indicate that the first strand exchange reaction is reversible during the integrative recombination.
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