Doctor of Philosophy (PHD)
Biochemistry and Molecular Biology (Medicine)
Date of Defense
First Committee Member
Walter A. Scott - Committee Chair
Second Committee Member
Kenneth E. Rudd - Committee Member
Third Committee Member
Vladlen Z. Slepak - Committee Member
Fourth Committee Member
Richard S. Myers - Mentor
Fifth Committee Member
Anthony Poteete - Outside Committee Member
In many dsDNA viruses, a single-strand-annealing homologous recombination (SSA) reaction is catalyzed by a pair of proteins. In phage lambda, this system is called Red, and is composed of lambda exonuclease (Lambda-Exo, a 5' to 3' dsDNA exonuclease), and Red-Beta (a ssDNA binding protein and annealase). I examined the physical and mechanistic coupling of Lambda-Exo and Red-Beta and confirmed that these proteins form a complex with a 1:1 subunit stoichiometry. The size of this complex was shown to be close to one MDa, possibly composed of 12 Lambda-Exo and 12 Red-Beta monomers. Red-Beta decreased the extent of digestion of dsDNA by Lambda-Exo, possibly by preventing its rebinding. The processivity of Lambda-Exo was not affected by Red-Beta, but the dwell-time of Lambda-Exo was significantly increased by Red-Beta. These effects of Red-Beta on Lambda-Exo may have important roles in controlling the SSA reaction by preventing hyper-resection of DNA, and/or by stabilizing Lambda-Exo/DNA complexes. The previous observations that Red-Beta protects ssDNA from nucleases and that SSB inhibits Red-Beta assembly onto ssDNA were confirmed and strengthened by our results. We determined that Red-Beta inhibits SSB binding to nascent ssDNA generated by Lambda-Exo. This strongly suggests that generation of nascent ssDNA by Lambda-Exo is coupled to assembly of Red-Beta onto nascent ssDNA. We describe two models for this coupled assembly: Model One suggests that Lambda-Exo loads Red-Beta on nascent ssDNA providing a kinetic advantage over SSB, and Model Two proposes that the complex of Lambda-Exo and Red-Beta feeds ssDNA directly onto the dodecameric Red-Beta ring. It was suggested that Lambda-Exo forms a topological link with nascent ssDNA, thereby making digestion highly processive. We challenged this model by removing the nascent ssDNA with ExoI during a Lambda-Exo digestion reaction. We observed that the nascent ssDNA was a major contributor to the processivity of Lambda-Exo since removal of nascent ssDNA resulted in a drastic decrease in the processivity of Lambda-Exo. This is the first demonstration that DNA is a processivity factor, strengthening the view that processive DNA processing enzymes should be thought of as nucleoprotein complexes that must be kinetically treated as both substrate and enzyme.
Tolun, Gokhan, "More than the Sum of Its Parts: Physical and Mechanistic Coupling in the Phage Lambda Red Recombinase" (2008). Open Access Dissertations. Paper 29.