Overexpression, purification, and characterization of recombinant T4 gene 32 protein22-301 (g32P-B).

Giedroc, D.P.; Khan, Raza; Barnhart, Kirstin

doi:10.1016/s0021-9258(19)38418-2

Cited by 47 publications

(48 citation statements)

References 39 publications

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“…The poly(A) used in these experiments was polydisperse, with the smallest fragments running along side of ∼500 bp duplex DNA fragment. Wild-type and B-domain mutant gp32s were expressed and purified essentially as described (Giedroc et al, 1990) with the modifications outlined in Villemain and Giedroc (1993). R4K, R4Q, R4T, R4G, and K3A gp32s refer respectively to Arg 4 fLys, Arg 4 fGln, Arg 4 fThr, Arg 4 fGly, and Lys 3 fAla single amino acid substitution mutants of wild-type gp32 (Villemain & Giedroc, 1993; gp32-B is an N-terminal truncation mutant which lacks residues 1-21 (Giedroc et al, 1990).…”

Section: Methodsmentioning

confidence: 99%

“…Wild-type and B-domain mutant gp32s were expressed and purified essentially as described (Giedroc et al, 1990) with the modifications outlined in Villemain and Giedroc (1993). R4K, R4Q, R4T, R4G, and K3A gp32s refer respectively to Arg 4 fLys, Arg 4 fGln, Arg 4 fThr, Arg 4 fGly, and Lys 3 fAla single amino acid substitution mutants of wild-type gp32 (Villemain & Giedroc, 1993; gp32-B is an N-terminal truncation mutant which lacks residues 1-21 (Giedroc et al, 1990). In general, the proteins used in this study were freshly thawed from frozen stocks and diluted with T buffer containing the desired [NaCl] before each experiment.…”

Section: Methodsmentioning

confidence: 99%

“…In general, the proteins used in this study were freshly thawed from frozen stocks and diluted with T buffer containing the desired [NaCl] before each experiment. The concentrations of all gp32s and poly(A) were determined from 280 ) 4.13 × 10 4 M -1 cm -1 (Giedroc et al, 1990) and 260 ) 10 300 M (nucleotide) -1 cm -1 (Kowalczykowski et al, 1981), respectively.…”

Section: Methodsmentioning

confidence: 99%

“…The primary structure of gp32 (301 amino acids) (Williams et al, 1980) consists of three domains as deduced by limited proteolysis experiments (Spicer et al, 1979). From studies of the binding of wild-type gp32 and gp32 truncation mutants (Kowalczykowski et al, 1981;Newport et al, 1981;Lonberg et al, 1981;Spicer et al, 1979;Giedroc et al, 1990), the N-terminal "B" or basic domain (residues 1-21) has been proposed to contain major determinants for cooperativity of binding, while the C-terminal "A" or acidic domain (residues 254-301) participates in heterologous protein-protein interactions essential for function of the multiprotein replication (Nossal, 1992;Hurley et al, 1993) and recombination complexes (Jiang et al, 1993;Salinas & Kodadek, 1995). The remainder of the protein is the core domain or fragment (residues 22-253) and contains an intrinsic Zn 2+ ion and major determinants for ssDNA binding.…”

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confidence: 99%

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The N-Terminal B-Domain of T4 Gene 32 Protein Modulates the Lifetime of Cooperatively Bound Gp32−ss Nucleic Acid Complexes

Villemain

Giedroc

1996

Biochemistry

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The N-terminal basic or B-domain (residues 1-21) of bacteriophage T4 gene 32 protein (gp32) provides a major determinant for highly cooperative binding by gp32 to single-stranded (ss) nucleic acids at equilibrium. In order to gain mechanistic insight into N-terminal domain function, the kinetics of dissociation of wild-type and previously characterized B-domain substitution mutant gp32s (R4K, R4Q, and K3A) from the model ribohomopolymer, poly(A), have been investigated under solution conditions identical to those used for equilibrium studies [Villemain, J. L., & Giedroc, D. P. (1993) Biochemistry 32, 11235-11246; Villemain, J. L., & Giedroc, D. P. (1996) J. Biol. Chem. 271, 27623-27629]. The dissociation of cooperatively bound gp32-poly(A) complexes was induced by sodium chloride concentration jumps and monitored by an increase in tryptophan fluorescence upon dissociation of the protein from poly(A) using stopped-flow techniques. The apparent dissociation rate constant, kd(app), for all mutant proteins studied was found to depend strongly on the initial fractional saturation of poly(A) just as was found previously for wild-type gp32. This permitted application of Lohman's model for the irreversible dissociation of cooperatively bound gp32-nucleic acid complexes [Lohman, T. M. (1983) Biopolymers 22, 1697-1713] from which the molecular rate constant, ke, the rate of dissociation of a protein monomer from teh end of a gp32-ss nucleic acid complex or protein cluster, could be determined. From the [NaCl]-dependence of kd(app), ke determined at 0.45 M NaCl, pH 8.1, 20 degrees C, was found to be 62 +/- 23, 78 +/- 8, 328 +/- 36, and 384 +/- 34 s-1 for wild-type, R4K, K3A, and R4Q gp32s, respectively. With the exception of R4K gp32, we find a striking correlation between the relative magnitudes of ke and Kapp, suggesting that the molecular defect in the equilibrium binding properties of the N-terminal domain mutants resides in the increased rate at which gp32 monomers dissociate from singly contiguous binding sites at the ends of clusters. The bimolecular association rate constant measured for wild-type gp32 and a weakly binding B-domain mutant, R4T gp32, to poly(dT) was found to be nearly identical, further evidence that the primary defect is in the dissociation reaction. We conclude that the N-terminal domain strongly modulates the lifetime of cooperatively bound gp32-polynucleotide complexes. The mechanistic and functional implications of these findings are discussed.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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The N-Terminal B-Domain of T4 Gene 32 Protein Modulates the Lifetime of Cooperatively Bound Gp32−ss Nucleic Acid Complexes

Villemain

Giedroc

1996

Biochemistry

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“…As a central player of the replisome, ssDNA binding protein (gp32) is likely to be involved in the initiation of lagging strand synthesis. Gp32 is made up of three domains: 31 the N-terminal domain (domain B for ''basic'') is involved in cooperative ssDNA binding, 32,33 the core domain is responsible for the recognition and binding of ssDNA, 34 and the C-terminal domain (domain A for ''acidic'') interacts with other T4 proteins. 35 We recently examined the relationship between the ssDNA binding protein, primase, clamp, and clamp loader during the initiation of Okazaki fragment synthesis by removing the protein interaction domain of gp32 (hereafter referred to as gp32-A) and observing the effect on primer synthesis, primer utilization, and primase processivity.…”

Section: Primer Handoffmentioning

confidence: 99%

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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Retroviral nucleocapsid proteins possess potent nucleic acid strand renaturation activity

1993

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The nucleocapsid protein (NC) is the major genomic RNA binding protein that plays integral roles in the structure and replication of all animal retroviruses. In this report, select biochemical properties of recombinant MasonPfizer monkey virus (MPMV) and HIV-1 NCs are compared. Evidence is presented that two types of saturated Zn, NC-polynucleotide complexes can be formed under conditions of low [NaCI] that differ in apparent site-size (n = 8 vs. n = 14). The formation of one or the other complex appears dependent on the molar ratio of NC to RNA nucleotide with the putative low site-size mode apparently predominating under conditions of protein excess. Both MPMV and HIV-1 NCs kinetically facilitate the renaturation of two complementary DNA strands, suggesting that this is a general property of retroviral NCs. NC proteins increase the second-order rate constant for renaturation of a 149-bp DNA fragment by more than four orders of magnitude over that obtained in the absence of protein at 37 "C. The protein-assisted rate is 100-200-fold faster than that obtained at 68 "C, 1 M NaCI, solution conditions considered t o be optimal for strand renaturation. Provided that sufficient NC is present to coat all strands, the presence of 400-1,000-fold excess nonhomologous DNA does not greatly affect the reaction rate. The HIV-1 NC-mediated renaturation reaction functions stoichiometrically, requiring a saturated strand of DNA nuc1eotide:NC ratio of about 7-8, rather than 14. Under conditions of less protein, the rate acceleration is not realized. The finding of significant nucleic acid strand renaturation activity may have important implications for various events of reverse transcription particularly in initiation and cDNA strand transfer.

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Overexpression, purification, and characterization of recombinant T4 gene 32 protein22-301 (g32P-B).

Cited by 47 publications

References 39 publications

The N-Terminal B-Domain of T4 Gene 32 Protein Modulates the Lifetime of Cooperatively Bound Gp32−ss Nucleic Acid Complexes

The N-Terminal B-Domain of T4 Gene 32 Protein Modulates the Lifetime of Cooperatively Bound Gp32−ss Nucleic Acid Complexes

Repetitive lagging strand DNA synthesis by the bacteriophage T4 replisome

Retroviral nucleocapsid proteins possess potent nucleic acid strand renaturation activity

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