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Mapping the interactions of the single-stranded DNA binding protein of bacteriophage T4 (gp32) with DNA lattices at single nucleotide resolution: gp32 monomer binding.

Jose D, Weitzel SE, Baase WA, von Hippel PH - Nucleic Acids Res. (2015)

Bottom Line: We show that single gp32 molecules interact most directly and specifically near the 3'-end of these ssDNA oligomers, thus defining the polarity of gp32 binding with respect to the ssDNA lattice, and that only 2-3 nts are directly involved in this tight binding interaction.The loss of exciton coupling in the CD spectra of dimer 2-AP (2-aminopurine) probes at various positions in the ssDNA constructs, together with increases in fluorescence intensity, suggest that gp32 binding directly extends the sugar-phosphate backbone of this ssDNA oligomer, particularly at the 3'-end and facilitates base unstacking along the entire 8-mer lattice.These results provide a model (and 'DNA map') for the isolated gp32 binding to ssDNA targets, which serves as the nucleation step for the cooperative binding that occurs at transiently exposed ssDNA sequences within the functioning T4 DNA replication complex.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, OR 97403-1229, USA.

No MeSH data available.


Related in: MedlinePlus

Stern–Volmer plots for fluorescence quenching by acrylamide of dT-containing ssDNA oligomers with 2-AP dimer probes at different positions in the absence (solid lines) and presence (dashed lines) of gp32 monomers. (A) 8P2,3 construct (3 μM free DNA, filled circles; plus 3 μM gp32, open circles; plus 6 μM gp32, open squares). (B) 8P4,5 construct (3 μM free DNA, filled circles; plus 3 μM gp32, open circles; plus 6 μM gp32, open squares). (C) 8P6,7 construct (3 μM free DNA, filled circles; plus 3 μM gp32, open circles; plus 6 μM gp32, open squares). Buffer and temperature conditions as in Figures 1 and 2.
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Figure 4: Stern–Volmer plots for fluorescence quenching by acrylamide of dT-containing ssDNA oligomers with 2-AP dimer probes at different positions in the absence (solid lines) and presence (dashed lines) of gp32 monomers. (A) 8P2,3 construct (3 μM free DNA, filled circles; plus 3 μM gp32, open circles; plus 6 μM gp32, open squares). (B) 8P4,5 construct (3 μM free DNA, filled circles; plus 3 μM gp32, open circles; plus 6 μM gp32, open squares). (C) 8P6,7 construct (3 μM free DNA, filled circles; plus 3 μM gp32, open circles; plus 6 μM gp32, open squares). Buffer and temperature conditions as in Figures 1 and 2.

Mentions: Acrylamide monomers in solution can serve as neutral collisional quenching agents, and their quenching efficiencies in different contexts depend on the accessibility of the 2-AP dimer probes to the aqueous solvent (29,32,41). Here acrylamide quenching was used to study the solvent accessibility of probe bases at various positions of our 8-mer ssDNA constructs in both the free and the gp32-bound states. Increasing concentrations of acrylamide were added to 3 μM concentrations of DNA constructs at 0, 3 or 6 μM concentrations of gp32 monomers, respectively, and the results are presented as Stern–Volmer plots in Figure 4.


Mapping the interactions of the single-stranded DNA binding protein of bacteriophage T4 (gp32) with DNA lattices at single nucleotide resolution: gp32 monomer binding.

Jose D, Weitzel SE, Baase WA, von Hippel PH - Nucleic Acids Res. (2015)

Stern–Volmer plots for fluorescence quenching by acrylamide of dT-containing ssDNA oligomers with 2-AP dimer probes at different positions in the absence (solid lines) and presence (dashed lines) of gp32 monomers. (A) 8P2,3 construct (3 μM free DNA, filled circles; plus 3 μM gp32, open circles; plus 6 μM gp32, open squares). (B) 8P4,5 construct (3 μM free DNA, filled circles; plus 3 μM gp32, open circles; plus 6 μM gp32, open squares). (C) 8P6,7 construct (3 μM free DNA, filled circles; plus 3 μM gp32, open circles; plus 6 μM gp32, open squares). Buffer and temperature conditions as in Figures 1 and 2.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4627070&req=5

Figure 4: Stern–Volmer plots for fluorescence quenching by acrylamide of dT-containing ssDNA oligomers with 2-AP dimer probes at different positions in the absence (solid lines) and presence (dashed lines) of gp32 monomers. (A) 8P2,3 construct (3 μM free DNA, filled circles; plus 3 μM gp32, open circles; plus 6 μM gp32, open squares). (B) 8P4,5 construct (3 μM free DNA, filled circles; plus 3 μM gp32, open circles; plus 6 μM gp32, open squares). (C) 8P6,7 construct (3 μM free DNA, filled circles; plus 3 μM gp32, open circles; plus 6 μM gp32, open squares). Buffer and temperature conditions as in Figures 1 and 2.
Mentions: Acrylamide monomers in solution can serve as neutral collisional quenching agents, and their quenching efficiencies in different contexts depend on the accessibility of the 2-AP dimer probes to the aqueous solvent (29,32,41). Here acrylamide quenching was used to study the solvent accessibility of probe bases at various positions of our 8-mer ssDNA constructs in both the free and the gp32-bound states. Increasing concentrations of acrylamide were added to 3 μM concentrations of DNA constructs at 0, 3 or 6 μM concentrations of gp32 monomers, respectively, and the results are presented as Stern–Volmer plots in Figure 4.

Bottom Line: We show that single gp32 molecules interact most directly and specifically near the 3'-end of these ssDNA oligomers, thus defining the polarity of gp32 binding with respect to the ssDNA lattice, and that only 2-3 nts are directly involved in this tight binding interaction.The loss of exciton coupling in the CD spectra of dimer 2-AP (2-aminopurine) probes at various positions in the ssDNA constructs, together with increases in fluorescence intensity, suggest that gp32 binding directly extends the sugar-phosphate backbone of this ssDNA oligomer, particularly at the 3'-end and facilitates base unstacking along the entire 8-mer lattice.These results provide a model (and 'DNA map') for the isolated gp32 binding to ssDNA targets, which serves as the nucleation step for the cooperative binding that occurs at transiently exposed ssDNA sequences within the functioning T4 DNA replication complex.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, OR 97403-1229, USA.

No MeSH data available.


Related in: MedlinePlus