Different DNA-binding modes and cooperativities for bacteriophage M13 gene-5 protein revealed by means of fluorescence depolarisation studies

Bulsink, Henk; Harmsen, B.J.M.; Hilbers, C.W.; Resandt, R. W. Wijnaendts van

doi:10.1111/j.1432-1033.1986.tb09672.x

Cited by 28 publications

(26 citation statements)

References 23 publications

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“…The filamentous bacteriophage Pfl gene 5 protein has two distinct modes of ssDNA binding which appear to result from the occupancy of either one or two DNA-binding sites on a gene 5 homodimer by ssDNA (11). Other SSB proteins have somewhat similar properties (10,29 (Fig. 8A).…”

Section: Resultsmentioning

confidence: 75%

Human replication protein A binds single-stranded DNA in two distinct complexes.

Blackwell¹,

Borowiec²

1994

Mol. Cell. Biol.

131

141

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Human replication protein A, a single-stranded DNA (ssDNA)-binding protein, is a required factor in eukaryotic DNA replication and DNA repair systems and has been suggested to function during DNA recombination. The protein is also a target of interaction for a variety of proteins that control replication, transcription, and cell growth. To understand the role of hRPA in these processes, we examined the binding of hRPA to defined ssDNA molecules. Employing gel shift assays that "titrated" the length of ssDNA, hRPA was found to form distinct multimeric complexes that could be detected by glutaraldehyde cross-linking. Within these complexes, monomers of hRPA utilized a minimum binding site size on ssDNA of 8 to 10 nucleotides (the hRPA8_10t complex) and appeared to bind ssDNA cooperatively. Intriguingly, alteration of gel shift conditions revealed the formation of a second, distinctly different complex that bound ssDNA in roughly 30-nucleotide steps (the hRPA30t complex), a complex similar to that described by Kim et al. (C. Kim, R. 0. Snyder, and M. S. Wold, Mol. Cell. Biol. 12:3050-3059, 1992). Both the hRPA8_l10t and hRPA30t complexes can coexist in solution. We speculate that the role of hRPA in DNA metabolism may be modulated through the ability of hRPA to bind ssDNA in these two modes.The replication, recombination, and repair of the cellular genetic information entail interconversion of DNA between the duplex and single-stranded forms. Study of nucleic acid enzymology has shown that single-stranded DNA (ssDNA) generated within cells is invariably associated with protein factors, often ssDNA-binding proteins (SSBs). SSBs combine with the ssDNA to form protein-DNA complexes that maintain the unwound state and are more active as substrates, for example, in DNA replication (12). A clear understanding of the architecture of SSB-ssDNA complexes is required to comprehend the function of SSB in DNA metabolism and determine how these processes are regulated within the cell.Eukaryotic SSBs with defined roles in DNA enzymology have recently been isolated, one of which, from human cells, is termed human replication protein A (hRPA). hRPA, a heterotrimer with subunits of 68, 29, and 14 kDa, was initially isolated as a factor from primate cells required to support simian virus 40 (SV40) DNA replication in vitro (24,42,43). The factor was identified as an SSB by various criteria including the high affinity of hRPA for ssDNA (24,42,43) and ability to stimulate human DNA polymerase a, an essential eukaryotic replication factor (22, 31). hRPA is phosphorylated in a cell-cycle specific manner, suggesting that DNA replication and other hRPA-mediated events may be modulated through regulation of hRPA activity (18,21,25). Moreover, recent studies have shown that hRPA specifically interacts with a number of transcription factors, suggesting that these factors regulate DNA replication, in part, through hRPA (20, 27, 35). In addition to its defined role in SV40 DNA replication, hRPA is also a required factor in an in vitro system...

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Section: Resultsmentioning

confidence: 75%

Human replication protein A binds single-stranded DNA in two distinct complexes.

Blackwell¹,

Borowiec²

1994

Mol. Cell. Biol.

131

141

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“…This loop includes residues [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] (Fig. 2 6 Harker peak height is the ratio of the value of the Patterson function at the lowest expected self-vector to the rms of the origin-removed map.…”

Section: Resultsmentioning

confidence: 99%

“…Gene V protein binding to single-stranded nucleic acids and to oligonucleotides has been studied using chemical modification, spectroscopic techniques, and mutagenesis (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). The structure of the protein-ssDNA complex has been studied using electron microscopy and solution scattering methods and is found to consist of a regular left-handed superhelix in which the gene V protein dimers are arrayed on the outside of the superhelix and the ssDNA strands are inside (8,25).…”

mentioning

confidence: 99%

Structure of the gene V protein of bacteriophage f1 determined by multiwavelength x-ray diffraction on the selenomethionyl protein.

Skinner

Zhang

Leschnitzer

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

105

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The crystal structure of the dimeric gene V protein of bacteriophage fl was determined using multiwavelength anomalous diffraction on the selenomethioninecontaining wild-type and isoleucine-47 methionine mutant proteins with x-ray diffraction data phased to 2.5 A resolution.The structure of the wild-type protein has been refined to an R factor of 19.2% using native data to 1.8 A resolution. The structure of the gene V protein was used to obtain a model for the protein portion of the gene V protein-single-stranded DNA complex.Gene V protein of bacteriophage fltt is a member of a class of proteins involved in DNA replication that bind to singlestranded nucleic acids with high affinity and cooperativity but little sequence specificity (1-4). Gene V protein coats the single-stranded DNA (ssDNA) intermediate in bacteriophage fl DNA replication, forming an ordered superhelical protein-DNA complex (1,(5)(6)(7)(8). This protein-DNA complex facilitates packaging of the ssDNA into new phage particles. Gene V protein also binds with some specificity to a translational operator sequence on phage fl gene II mRNA (9-12).The gene V protein provides a general model for proteinssDNA interactions that are strong, yet not sequencespecific. Gene V protein binding to single-stranded nucleic acids and to oligonucleotides has been studied using chemical modification, spectroscopic techniques, and mutagenesis (13-24). The structure of the protein-ssDNA complex has been studied using electron microscopy and solution scattering methods and is found to consist of a regular left-handed superhelix in which the gene V protein dimers are arrayed on the outside of the superhelix and the ssDNA strands are inside (8,25). The structure of the gene V protein is also of interest because its small size and the large number of mutants available have made it a useful model for determining effects ofamino acid substitutions on protein stability and function (23,26,27).A model for the crystal structure of the wild-type (WT) gene V protein has been reported (28), but recent NMR studies have demonstrated that the positions of amino acids involved in the segments of antiparallel (-structure are not compatible with those in the model (21), and a new determination of the structure was necessary. The multiwavelength anomalous diffraction (MAD) technique (29) was ideally suited for this purpose, as the WT gene V protein contains two methionine residues (Met-1 and Met-77) that might be substituted in vivo in Escherichia coli by selenomethionine. Here we report the determination of the gene V protein structure using the MAD technique, the refinement of the structure using x-ray diffraction data on the WT gene V proteinAt and a model for protein-protein contacts in the gene V protein-ssDNA superhelical complex. MATERIALS AND METHODSModeling of the Protein Portion of Gene V Protein-ssDNA Complex. The gene V protein dimer was placed with its internal two-fold axis of symmetry perpendicular to the axis of the superhelix to be generated and Phe-73 pointing ei...

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“…The presence of a dominant slow anisotropy decay component (Table 3) undoubtedly represented the tumbling of the entire molecule, because the rotational correlation time of the g5p dimer is 12.9 ns. 50 We conclude from the anisotropy results that the various states of 2AP sequestered within the g5p binding site represented relatively inflexible conformations.…”

Section: Biochemistrymentioning

confidence: 95%

Ultrafast Fluorescence Decay Profiles Reveal Differential Unstacking of 2-Aminopurine from Neighboring Bases in Single-Stranded DNA-Binding Protein Subsites

et al. 2011

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Gene 5 protein (g5p) is a dimeric single-stranded DNA-binding protein encoded by Ff strains of Escherichia coli bacteriophages. The 2-fold rotationally symmetric binding sites of a g5p dimer each bind to four nucleotides, and the dimers bind with high cooperativity to saturate antiparallel single-stranded DNA (ssDNA) strands. Ultrafast time-resolved fluorescence spectroscopies were used to investigate the conformational heterogeneity and dynamics of fluorescent 2-aminopurine (2AP) labels sequestered by bound g5p. The 2AP labels were positioned within the noncomplementary antiparallel tail sequences of d(AC)(8) or d(AC)(9) of hairpin constructs so that each fluorescent label could probe a different subsite location within the DNA-binding site of g5p. Circular dichroism and isothermal calorimetric titrations yielded binding stoichiometries of approximately six dimers per oligomer hairpin when tails were of these lengths. Mobility shift assays demonstrated the formation of a single type of g5p-saturated complex. Femtosecond time-resolved fluorescence spectroscopy showed that the 2AP in the free (non-protein-bound) DNAs had similar heterogeneous distributions of conformations. However, there were significant changes, dominated by a large increase in the population of unstacked bases from ~22 to 59-68%, depending on their subsite locations, when the oligomers were saturated with g5p. Anisotropy data indicated that 2AP in the bound state was less flexible than in the free oligomer. A control oligomer was labeled with 2AP in the loop of the hairpin and showed no significant change in its base stacking upon g5p binding. A proposed model summarizes the data.

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Different DNA-binding modes and cooperativities for bacteriophage M13 gene-5 protein revealed by means of fluorescence depolarisation studies

Cited by 28 publications

References 23 publications

Human replication protein A binds single-stranded DNA in two distinct complexes.

Human replication protein A binds single-stranded DNA in two distinct complexes.

Structure of the gene V protein of bacteriophage f1 determined by multiwavelength x-ray diffraction on the selenomethionyl protein.

Ultrafast Fluorescence Decay Profiles Reveal Differential Unstacking of 2-Aminopurine from Neighboring Bases in Single-Stranded DNA-Binding Protein Subsites

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