Assembly and Dynamics of Gp59-Gp32-Single-stranded DNA (ssDNA), a DNA Helicase Loading Complex Required for Recombination-dependent Replication in Bacteriophage T4

Branagan, Amy; Maher, Robyn L.; Morrical, Scott W.

doi:10.1074/jbc.m112.343830

Cited by 5 publications

(23 citation statements)

References 44 publications

(69 reference statements)

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“…3A and 3B. Effect of I87A Mutation on HLC Remodeling during Helicase Loading on ssDNA-It was previously shown through Gp32F fluorescence assays that the loading of Gp41 helicase remodels the HLC and that this remodeling is dependent on ATP binding (but not hydrolysis) by Gp41 (20) (Fig. 4A).…”

Section: Comparison Of Dna Binding Activities Of Gp59 Wt and I87a-mentioning

confidence: 85%

“…We have also shown that the fluorescence intensity of Gp32F is increased when it is ssDNA-bound and further increased when it is in a helicase loading complex with ssDNA and Gp59 (20,23). This Gp32F fluorescence assay was used to determine the effects of the Gp59-I87A mutation on HLC formation (Fig.…”

Section: Comparison Of Dna Binding Activities Of Gp59 Wt and I87a-mentioning

confidence: 93%

“…Gp32, Gp41, and Gp59 proteins were purified as described previously (14,20). Gp43-D219A, an exonuclease-deficient form of the Gp43 T4 polymerase, was a gift from Dr. Linda Reha-Krantz (University of Alberta).…”

Section: Methodsmentioning

confidence: 99%

“…Fluorescence Assays for Assembly and Helicase Loading Activity of Helicase Loading Complexes-Changes in Gp32F fluorescence were monitored to detect HLC formation on ssDNA and its remodeling upon helicase loading, as previously described (20). Reactions were carried out at room temperature.…”

Section: Methodsmentioning

confidence: 99%

“…However, under physiological conditions during RDR, Gp59 most likely co-occupies ssDNA with Gp32 to form a tripartite helicase loading complex (HLC) in which the DNA binding properties of both proteins are altered (19). Additionally, we recently provided evidence that Gp32 clusters on ssDNA may be the target for Gp59 binding rather than DNA itself and that Gp32 and Gp59 likely remain bound to each other throughout T4 RDR (20). Based on these observations, we hypothesize that helicase loading in RDR may depend more on Gp59-Gp32 interactions than on Gp59-DNA interactions.…”

mentioning

confidence: 99%

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Control of Helicase Loading in the Coupled DNA Replication and Recombination Systems of Bacteriophage T4

Branagan

Klein

Jordan

et al. 2014

Journal of Biological Chemistry

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Background: Helicase loading protein Gp59 coordinates recombination-dependent DNA replication in bacteriophage T4. Results: A DNA binding-deficient mutant, Gp59-I87A, loads helicase normally onto ssDNA covered with the ssDNA-binding protein, Gp32. Conclusion: Gp59-Gp32 interactions control helicase loading onto D-loops during recombination-dependent replication. Significance: Helicase loading proteins work in concert with single-stranded DNA binding proteins to control the location and timing of helicase assembly in DNA replication, recombination, and repair.

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Section: Comparison Of Dna Binding Activities Of Gp59 Wt and I87a-mentioning

confidence: 85%

Section: Comparison Of Dna Binding Activities Of Gp59 Wt and I87a-mentioning

confidence: 93%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Control of Helicase Loading in the Coupled DNA Replication and Recombination Systems of Bacteriophage T4

Branagan

Klein

Jordan

et al. 2014

Journal of Biological Chemistry

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Regulation of the bacteriophage T4 Dda helicase by Gp32 single-stranded DNA–binding protein

Jordan

Morrical

2015

DNA Repair

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Dda, one of three helicases encoded by bacteriophage T4, has been well-characterized biochemically but its biological role remains unclear. It is thought to be involved in origin dependent DNA replication, recombination-dependent replication, anti-recombination, and recombination repair. The Gp32 protein of bacteriophage T4 plays critical roles in DNA replication, recombination, and repair by coordinating protein components of the replication fork and by stabilizing ssDNA. Previous work demonstrated that stimulation of DNA synthesis by Dda helicase appears to require direct Gp32-Dda protein-protein interactions and that Gp32 and Dda form a tight complex in the absence of ssDNA. Here we characterize the effects of Gp32-Dda physical and functional interactions through changes in the duplex DNA unwinding and ATPase activities of Dda helicase in the presence of different variants of Gp32 and different DNA repair and replication intermediate structures. Results show that Gp32-Dda interactions can be enhancing or inhibitory, depending on the Gp32 domain seen by Dda. Protein-protein interactions with Gp32 stimulate the unwinding activity of Dda, an effect associated with increased turnover of ATP, suggesting a higher rate of ATPase-driven translocation. Dda-Gp32 interactions also promote the unwinding of DNA substrates at higher salt concentrations and in the presence of substrate-bound DNA polymerase. Conversely, the formation of Gp32 clusters on ssDNA can inhibit unwinding, suggesting that Gp32-ssDNA formation sterically regulates which portions of replication and recombination intermediates are accessible for processing by Dda helicase. The data suggest a mechanism of replication fork restart in which Gp32 promotes Dda activity in template switching while preventing premature fork progression.

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Interaction of T4 UvsW Helicase and Single-Stranded DNA Binding Protein gp32 through Its Carboxy-Terminal Acidic Tail

Perumal¹,

Nelson²,

Benkovic³

2013

Journal of Molecular Biology

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Bacteriophage T4 UvsW helicase contains both unwinding and annealing activities and displays some functional similarities to bacterial RecG and RecQ helicases. UvsW is involved in several DNA repair pathways, playing important roles in recombination-dependent DNA repair and the reorganization of stalled replication forks. The T4 single-stranded DNA binding protein, gp32, is a central player in nearly all DNA replication and repair processes and is thought to facilitate their coordination by recruiting and regulating the various proteins involved. Here, we show that the activities of the UvsW protein are modulated by gp32. UvsW catalyzed unwinding of recombination intermediates such as D-loops and static X-DNA (Holliday junction mimic) to ssDNA products is enhanced by the gp32 protein. The enhancement requires the presence of the protein interaction domain of gp32 (the acidic carboxy terminus), suggesting that a specific interaction between UvsW and gp32 is required. In the absence of this interaction, the ssDNA annealing and ATP-dependent translocation activities of UvsW are severely inhibited when gp32 coats the ssDNA lattice. However, when UvsW and gp32 do interact, UvsW is able to efficiently displace the gp32 protein from the ssDNA. This ability of UvsW to remove gp32 from ssDNA may explain its ability to enhance the strand invasion activity of the T4 recombinase (UvsX) and suggests a possible new role for UvsW in gp32-mediated DNA transactions.

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Assembly and Dynamics of Gp59-Gp32-Single-stranded DNA (ssDNA), a DNA Helicase Loading Complex Required for Recombination-dependent Replication in Bacteriophage T4

Cited by 5 publications

References 44 publications

Control of Helicase Loading in the Coupled DNA Replication and Recombination Systems of Bacteriophage T4

Control of Helicase Loading in the Coupled DNA Replication and Recombination Systems of Bacteriophage T4

Regulation of the bacteriophage T4 Dda helicase by Gp32 single-stranded DNA–binding protein

Interaction of T4 UvsW Helicase and Single-Stranded DNA Binding Protein gp32 through Its Carboxy-Terminal Acidic Tail

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