UVM Theses and Dissertations
Format:
Print
Author:
Branagan, Amy M.
Dept./Program:
Biochemistry
Year:
2012
Degree:
PhD
Abstract:
The repair of double strand DNA breaks (DSBs) is essential for the maintenance of genome stability. Homology-directed repair (HDR) is an evolutionarily conserved mechanism to correct this type of DNA damage. HDR is based on the use of an intact double stranded DNA segment" (usually a homologous chromosome) as a template to repair the broken DNA. In Bacteriophage T4, the majority of genome replication occurs by a similar mechanism, recombination-dependent replication (RDR), which makes it a particularly useful model system for the study of DSB repair. T4 is also a highly efficient, minimalist DNA replication system that can be reconstituted in vitro. For this thesis, we have used T4 to study an essential step in HDR that is conserved among organisms: helicase loading.
Helicase loading is a rate-limiting step in the transition of a recombination intermediate into a replication fork. It usually requires mediator proteins to assemble a DNA helicase onto fork DNA, which is often already bound by single stranded DNA binding proteins (SSBs). The focus of this thesis project is on the mechanisms of helicase loading by Gp59, a mediator protein in the Bacteriophage T4 system. In addition to its helicase loading function, we investigated the role of Gp59 as a polymerase blocking protein. This second function is necessary in order to mediate a polymerase/helicase functional interaction and to coordinate DNA synthesis on the leading and lagging strands.
In Chapter 2, we present our findings on the assembly and dynamics of the helicase loading complex (HLC). The HLC is a Gp32-ssDNA-Gp59 tripartite complex that is optimal for helicase loading. We find that Gp32 clusters act as a target for HLC formation on ssDNA; and that Gp32/Gp59 interactions are a critical factor in HLC assembly. Additionally, we find evidence that Gp59 may have the ability to carry out multiple cycles of helicase loading by re-binding to ssDNA. This has important implications for potentially re-starting replication forks and bypassing DNA lesions during replication.
In Chapter 3, we investigate the roles of Gp59/DNA interactions during T4 replication. Consistent with our findings in Chapter 2, we conclude that Gp59/DNA interactions are non-essential for helicase loading on Gp32-covered ssDNA, suggesting that this function may depend primarily on a protein/protein interaction with Gp32. Additionally, we conclude that polymerase blocking may depend primarily on a protein/protein interaction with Gp43. In contrast, we find Gp59/DNA interactions are essential for stimulating helicase unwinding of fork DNA substrates. The results presented in Chapter 3 lead us to conclude that Gp59 may require different binding interactions on ssDNA vs. fork DNA. This has significant implications for the differing roles of Gp59 in origin-dependent replication, initial replisome assembly during RDR, and replication fork restart. Our results have added to our knowledge of genome maintenance in T4 by clarifying the ways in which Gp59 helps to coordinate DNA replication. The work presented here should provide the basis for further experimentation of heIicase loading in more complex systems.
Helicase loading is a rate-limiting step in the transition of a recombination intermediate into a replication fork. It usually requires mediator proteins to assemble a DNA helicase onto fork DNA, which is often already bound by single stranded DNA binding proteins (SSBs). The focus of this thesis project is on the mechanisms of helicase loading by Gp59, a mediator protein in the Bacteriophage T4 system. In addition to its helicase loading function, we investigated the role of Gp59 as a polymerase blocking protein. This second function is necessary in order to mediate a polymerase/helicase functional interaction and to coordinate DNA synthesis on the leading and lagging strands.
In Chapter 2, we present our findings on the assembly and dynamics of the helicase loading complex (HLC). The HLC is a Gp32-ssDNA-Gp59 tripartite complex that is optimal for helicase loading. We find that Gp32 clusters act as a target for HLC formation on ssDNA; and that Gp32/Gp59 interactions are a critical factor in HLC assembly. Additionally, we find evidence that Gp59 may have the ability to carry out multiple cycles of helicase loading by re-binding to ssDNA. This has important implications for potentially re-starting replication forks and bypassing DNA lesions during replication.
In Chapter 3, we investigate the roles of Gp59/DNA interactions during T4 replication. Consistent with our findings in Chapter 2, we conclude that Gp59/DNA interactions are non-essential for helicase loading on Gp32-covered ssDNA, suggesting that this function may depend primarily on a protein/protein interaction with Gp32. Additionally, we conclude that polymerase blocking may depend primarily on a protein/protein interaction with Gp43. In contrast, we find Gp59/DNA interactions are essential for stimulating helicase unwinding of fork DNA substrates. The results presented in Chapter 3 lead us to conclude that Gp59 may require different binding interactions on ssDNA vs. fork DNA. This has significant implications for the differing roles of Gp59 in origin-dependent replication, initial replisome assembly during RDR, and replication fork restart. Our results have added to our knowledge of genome maintenance in T4 by clarifying the ways in which Gp59 helps to coordinate DNA replication. The work presented here should provide the basis for further experimentation of heIicase loading in more complex systems.