UVM Theses and Dissertations
Format:
Print
Author:
Bleuit, Jill Susan
Dept./Program:
Cell and Molecular Biology Program
Year:
2004
Degree:
Ph. D.
Abstract:
Homologous recombination is a fundamental process in all organisms. The basic mechanism has been well conserved from bacteria through humans, as an effective means of repairing double strand breaks in DNA and fostering genetic diversity. The initiation stage of homologous recombination involves processing a double-strand break into a substrate with a 3' single-strand DNA tail that is suitable for recognition by the proteins that catalyze recombination. These recombinase proteins then assemble onto the 3' tail to form a nucleoprotein filament, which is the active species for instigating strand exchange with a DNA molecule of homologous sequence. The bacteriophage T4 is a convenient model organism for studying recombination since, firstly, it uses a recombination-dependent replication pathway as its major means of reproduction, and secondly, most of the proteins involved in phage recombination have been identified and well characterized. An exception is the T4 protein(s) responsible for the initial processing step. Ample genetic evidence suggests that the Genes 46 and 47 encode a deoxyribonuclease that is essential for recombination, but the protein products of these genes, Gp46 and Gp47, respectively, have remained poorly characterized biochemically prior to this study.
This document describes the purification and first direct biochemical characterization of these proteins. My results suggest that the Gp46 protein is a 5' to 3' dsDNA exonuclease with endonuclease activjty as well, and that the Gp47 protein interacts with it and stimulates its nuclease activities. The preference of the Gp46/ Gp47 complex for degrading linear dsDNA ends, as well as it's 5' to 3' polarity, make it an excellent candidate for the initial resection of the 5' DNA strand at the outset of recombination. Once a 3' ssDNA tail has been exposed by the resection, a presynaptic filament of UvsX, the T4 recombinase, must be assembled on it. However, in order for this to happen, the UvsX must win a competition with Gp32, the T4 single-strand binding protein. Gp32 binds all available ssDNA to protect it from forming inhibitory secondary structures and from unprogrammed nucleolytic degradation. It is an abundant protein and has a stronger affinity for ssDNA than does UvsX. The UvsY protein, a member of a conserved class of proteins known as Recombination-Mediator Proteins, enables UvsX to replace Gp32 on ssDNA, and the intricacies of the mechanism it uses to do this are progressively being elucidated.
UvsY helps UvsX compete with Gp32 for binding sites on ssDNA, in part by destabilizing Gp32-ssDNA interactions, and in part by stabilizing UvsX-ssDNA interactions. The relative contributions of UvsY-ssDNA, UvsY-Gp32, UvsY-UvsX, and UvsY-UvsY interactions to these processes are only partially understood. Therefore, two UvsY mutant proteins were engineered that are deficient in ssDNA binding activity. These mutant proteins strongly inhibit, but do not completely eliminate, UvsY stimulation of UvsX-catalyzed reactions including ssDNA-dependent ATP hydrolysis and DNA strand exchange, both activities of which are contingent on the assembly of UvsX onto ssDNA under conditions that are made prohibitive by Gp32. A possible mechanism wherein UvsY displaces Gp32 by wrapping the DNA around it, breaking the cooperative interactions between Gp32 monomers, while simultaneously loading UvsX with its protein/ protein contacts, will be discussed. Also, intriguingly, both the Gp46 and Gp47 proteins were observed to interact with UvsY, which has implications for coupling nucleolytic processing of double-strand ends to presynaptic filament assembly. The journey of the DNA from double-stranded end to D-loop intermediate, and finally to resolved, recombinant copy, involves an elegant, and controlled series of interactions negotiated between the DNA and the various proteins of the recombination machine, that seem to pass intermediates along, hand to hand, until the DNA product is successfully constructed.
This document describes the purification and first direct biochemical characterization of these proteins. My results suggest that the Gp46 protein is a 5' to 3' dsDNA exonuclease with endonuclease activjty as well, and that the Gp47 protein interacts with it and stimulates its nuclease activities. The preference of the Gp46/ Gp47 complex for degrading linear dsDNA ends, as well as it's 5' to 3' polarity, make it an excellent candidate for the initial resection of the 5' DNA strand at the outset of recombination. Once a 3' ssDNA tail has been exposed by the resection, a presynaptic filament of UvsX, the T4 recombinase, must be assembled on it. However, in order for this to happen, the UvsX must win a competition with Gp32, the T4 single-strand binding protein. Gp32 binds all available ssDNA to protect it from forming inhibitory secondary structures and from unprogrammed nucleolytic degradation. It is an abundant protein and has a stronger affinity for ssDNA than does UvsX. The UvsY protein, a member of a conserved class of proteins known as Recombination-Mediator Proteins, enables UvsX to replace Gp32 on ssDNA, and the intricacies of the mechanism it uses to do this are progressively being elucidated.
UvsY helps UvsX compete with Gp32 for binding sites on ssDNA, in part by destabilizing Gp32-ssDNA interactions, and in part by stabilizing UvsX-ssDNA interactions. The relative contributions of UvsY-ssDNA, UvsY-Gp32, UvsY-UvsX, and UvsY-UvsY interactions to these processes are only partially understood. Therefore, two UvsY mutant proteins were engineered that are deficient in ssDNA binding activity. These mutant proteins strongly inhibit, but do not completely eliminate, UvsY stimulation of UvsX-catalyzed reactions including ssDNA-dependent ATP hydrolysis and DNA strand exchange, both activities of which are contingent on the assembly of UvsX onto ssDNA under conditions that are made prohibitive by Gp32. A possible mechanism wherein UvsY displaces Gp32 by wrapping the DNA around it, breaking the cooperative interactions between Gp32 monomers, while simultaneously loading UvsX with its protein/ protein contacts, will be discussed. Also, intriguingly, both the Gp46 and Gp47 proteins were observed to interact with UvsY, which has implications for coupling nucleolytic processing of double-strand ends to presynaptic filament assembly. The journey of the DNA from double-stranded end to D-loop intermediate, and finally to resolved, recombinant copy, involves an elegant, and controlled series of interactions negotiated between the DNA and the various proteins of the recombination machine, that seem to pass intermediates along, hand to hand, until the DNA product is successfully constructed.