Mutations in the N-terminal Cooperativity Domain of Gene 32 Protein Alter Properties of the T4 DNA Replication and Recombination Systems

Villemain, Jana L.; Ma, Yujie; Giedroc, D.P.; Morrical, Scott W.

doi:10.1074/jbc.m002902200

Cited by 11 publications

(15 citation statements)

References 36 publications

(46 reference statements)

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“…The hierarchy of activities obtained with missense mutants R4K, R4Q, K3A, R4T, and R4G in Fig. 2A is similar to that observed previously in strand displacement synthesis reactions performed in the absence of a DNA helicase (29).…”

Section: Effects Of Gp32 B-domain Missense Mutations On Strand Displasupporting

confidence: 66%

“…3A, lanes 2 and 4), dramatically shorter and less intense than wt in reactions with R4Q and K3A gp32s (lanes 5 and 7) and nonexistent in reactions with R4T and R4G gp32s (lanes 8 and 9). The hierarchy of gp32 missense mutant activities in the helicase minus fraction of these reactions matches precisely the hierarchy observed in reactions lacking gp41 entirely (29). Third, the only replication product visible in the reaction with gp32-B is the chew-back/fill-in product (Fig.…”

Section: Distribution Of Strand Displacement Synthesis Products As Asupporting

confidence: 49%

“…2C). This despite the fact that gp32-B binds very weakly and non-cooperatively to ssDNA and cannot saturate the lattice under our reaction conditions (29). The common thread between these six gp32 mutant species (R4K, R4Q, K3A, R4T, R4G, gp32-B) is that despite their drastic differences in ssDNA binding affinity, all retain protein-protein interactions with the dda helicase.…”

Section: Figmentioning

confidence: 96%

“…Strand Displacement DNA Synthesis-On a nicked template strand displacement DNA synthesis by T4 DNA polymerase holoenzyme requires the tight and cooperative binding of gp32 to the displaced ssDNA (29). Mutations in the B domain of gp32 that decrease the stability of gp32-ssDNA cooperative clusters also dramatically decrease the rate and processivity of strand displacement DNA synthesis (29).…”

Section: Relationship Between Equilibrium and Kinetic Properties Of Gmentioning

confidence: 99%

“…2 We previously showed that the ability of different gp32 B domain mutants to support strand displacement DNA synthesis (in the absence of a helicase) correlates strongly with their hierarchy of relative ssDNA binding affinities, i.e. gp32 wild type Ϸ R4K Ͼ K3A Ϸ R4Q Ͼ R4T Ͼ R4G Ͼ Ͼ gp32-B (29).…”

mentioning

confidence: 99%

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Dual Functions of Single-stranded DNA-binding Protein in Helicase Loading at the Bacteriophage T4 DNA Replication Fork

Wang

Villemain

et al. 2004

Journal of Biological Chemistry

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Semi-conservative DNA synthesis reactions catalyzed by the bacteriophage T4 DNA polymerase holoenzyme are initiated by a strand displacement mechanism requiring gp32, the T4 single-stranded DNA (ssDNA)-binding protein, to sequester the displaced strand. After initiation, DNA helicase acquisition by the nascent replication fork leads to a dramatic increase in the rate and processivity of leading strand DNA synthesis. In vitro studies have established that either of two T4-encoded DNA helicases, gp41 or dda, is capable of stimulating strand displacement synthesis. The acquisition of either helicase by the nascent replication fork is modulated by other protein components of the fork including gp32 and, in the case of the gp41 helicase, its mediator/ loading protein gp59. Here, we examine the relationships between gp32 and the gp41/gp59 and dda helicase systems, respectively, during T4 replication using altered forms of gp32 defective in either protein-protein or protein-ssDNA interactions. We show that optimal stimulation of DNA synthesis by gp41/gp59 helicase requires gp32-gp59 interactions and is strongly dependent on the stability of ssDNA binding by gp32. Fluorescence assays demonstrate that gp59 binds stoichiometrically to forked DNA molecules; however, gp59-forked DNA complexes are destabilized via protein-protein interactions with the C-terminal "A-domain" fragment of gp32. These and previously published results suggest a model in which a mobile gp59-gp32 cluster bound to lagging strand ssDNA is the target for gp41 helicase assembly. In contrast, stimulation of DNA synthesis by dda helicase requires direct gp32-dda protein-protein interactions and is relatively unaffected by mutations in gp32 that destabilize its ssDNA binding activity. The latter data support a model in which protein-protein interactions with gp32 maintain dda in a proper active state for translocation at the replication fork. The relationship between dda and gp32 proteins in T4 replication appears similar to the relationship observed between the UL9 helicase and ICP8 ssDNA-binding protein in herpesvirus replication.DNA helicase acquisition by a nascent DNA replication fork is essential for reconstituting rapid, processive DNA synthesis. This principle is illustrated dramatically by the bacteriophage T4 DNA replication system (1-2). T4 encodes two DNA helicases known to affect the movement and properties of DNA replication forks; the gene 41 protein (gp41) and the dda protein, respectively. gp41 is the essential replicative helicase of the T4 phage. This hexameric enzyme translocates processively in a 5Ј 3 3Ј direction on the displaced lagging strand of the dsDNA 1 template, thus enhancing the rate and processivity of leading strand DNA synthesis catalyzed by the T4 DNA polymerase holoenzyme (gp43, gp44/62, and gp45 proteins) (1-3). In addition, gp41 is an obligatory component of the T4 primosome (helicase/primase complex) and is, thus, essential for lagging strand DNA synthesis (4 -5). A second T4-encoded DNA helicase, dda protein, stimula...

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Section: Effects Of Gp32 B-domain Missense Mutations On Strand Displasupporting

confidence: 66%

Section: Distribution Of Strand Displacement Synthesis Products As Asupporting

confidence: 49%

Section: Figmentioning

confidence: 96%

Section: Relationship Between Equilibrium and Kinetic Properties Of Gmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Dual Functions of Single-stranded DNA-binding Protein in Helicase Loading at the Bacteriophage T4 DNA Replication Fork

Wang

Villemain

et al. 2004

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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Repetitive lagging strand DNA synthesis by the bacteriophage T4 replisome

2008

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Our studies on the T4 replisome build on the seminal work from the Alberts laboratory. They discovered essentially all the proteins that constitute the T4 replisome, isolated them, and measured their enzymatic activities. Ultimately, in brilliant experiments they reconstituted in vitro a functioning replisome and in the absence of structural information created a mosaic as to how such a machine might be assembled. Their consideration of the problem of continuous leading strand synthesis opposing discontinuous lagging strand synthesis led to their imaginative proposal of the trombone model, an illustration that graces all textbooks of biochemistry. Our subsequent work deepens their findings through experiments that focus on defining the kinetics, structural elements, and protein-protein contacts essential for replisome assembly and function. In this highlight we address when Okazaki primer synthesis is initiated and how the primer is captured by a recycling lagging strand polymerase--problems that the Alberts laboratory likewise found mysterious and significant for all replisomes.

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Mutations in the N-terminal Cooperativity Domain of Gene 32 Protein Alter Properties of the T4 DNA Replication and Recombination Systems

Cited by 11 publications

References 36 publications

Dual Functions of Single-stranded DNA-binding Protein in Helicase Loading at the Bacteriophage T4 DNA Replication Fork

Dual Functions of Single-stranded DNA-binding Protein in Helicase Loading at the Bacteriophage T4 DNA Replication Fork

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

Repetitive lagging strand DNA synthesis by the bacteriophage T4 replisome

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