Limits...
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

A model for the binding of single gp32 molecules to short ssDNA lattices. (A) Representation of the structure and electrostatic surface potential of the core domain of gp32; blue regions are basic and red regions are acidic (12). (B) The core gp32 domain with a bound ssDNA d(T)4 oligonucleotide fitted to the difference electron density (12). (C) Model of an isolated gp32 molecule showing the C-terminal arm tightly bound to the electropositive cleft of the core domain in the absence of ssDNA. (D) Model of the core gp32 domain bound to an 8-mer ssDNA lattice. The absence of the C-terminal flap allows the oligonucleotide to ‘shuffle’ along the protein lattice. (E) Model of the binding of a gp32 monomer to an 8-mer oligonucleotide. The positively charged binding cleft of the core domain is occluded by the C-terminal arm, thereby hindering the access of the oligonucleotide and resulting in more base unstacking at the 3′ end and less at the 5′ end of the 8-mer ssDNA lattice. This is represented by the decreasing gradient of chain extension from the 3′ to the 5′-end of the ssDNA oligomer. We suggest that this binding mode is likely to be in dynamic equilibrium with a gp32 monomer conformation (F) in which the C-terminal arm is flipped out of the positive binding cleft and binds to the previously proposed anion binding site (14,15) on the ‘top’ of the gp32 monomer, displacing bound monovalent anions there and permitting full oligonucleotide access to the gp32 binding cleft. Panels (A) and (B) of this figure have been reproduced, with permission, from (12).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4627070&req=5

Figure 6: A model for the binding of single gp32 molecules to short ssDNA lattices. (A) Representation of the structure and electrostatic surface potential of the core domain of gp32; blue regions are basic and red regions are acidic (12). (B) The core gp32 domain with a bound ssDNA d(T)4 oligonucleotide fitted to the difference electron density (12). (C) Model of an isolated gp32 molecule showing the C-terminal arm tightly bound to the electropositive cleft of the core domain in the absence of ssDNA. (D) Model of the core gp32 domain bound to an 8-mer ssDNA lattice. The absence of the C-terminal flap allows the oligonucleotide to ‘shuffle’ along the protein lattice. (E) Model of the binding of a gp32 monomer to an 8-mer oligonucleotide. The positively charged binding cleft of the core domain is occluded by the C-terminal arm, thereby hindering the access of the oligonucleotide and resulting in more base unstacking at the 3′ end and less at the 5′ end of the 8-mer ssDNA lattice. This is represented by the decreasing gradient of chain extension from the 3′ to the 5′-end of the ssDNA oligomer. We suggest that this binding mode is likely to be in dynamic equilibrium with a gp32 monomer conformation (F) in which the C-terminal arm is flipped out of the positive binding cleft and binds to the previously proposed anion binding site (14,15) on the ‘top’ of the gp32 monomer, displacing bound monovalent anions there and permitting full oligonucleotide access to the gp32 binding cleft. Panels (A) and (B) of this figure have been reproduced, with permission, from (12).

Mentions: Inspection of the crystal structure of the core DNA-binding domain of gp32 (Figure 6A) suggests that this decrease in access to collisional quenchers near the 3′-end of the 8-mer ssDNA constructs might reflect partial intercalation into the ssDNA chain at these positions of tyrosine side-chains within the DNA binding cleft (see (12)), which could be an additional determinant of the proposed backbone twisting and chain extension resulting from the tight binding of the gp32 to the sugar-phosphate backbones of the ssDNA chain near the 3′-end of the test oligomers.


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)

A model for the binding of single gp32 molecules to short ssDNA lattices. (A) Representation of the structure and electrostatic surface potential of the core domain of gp32; blue regions are basic and red regions are acidic (12). (B) The core gp32 domain with a bound ssDNA d(T)4 oligonucleotide fitted to the difference electron density (12). (C) Model of an isolated gp32 molecule showing the C-terminal arm tightly bound to the electropositive cleft of the core domain in the absence of ssDNA. (D) Model of the core gp32 domain bound to an 8-mer ssDNA lattice. The absence of the C-terminal flap allows the oligonucleotide to ‘shuffle’ along the protein lattice. (E) Model of the binding of a gp32 monomer to an 8-mer oligonucleotide. The positively charged binding cleft of the core domain is occluded by the C-terminal arm, thereby hindering the access of the oligonucleotide and resulting in more base unstacking at the 3′ end and less at the 5′ end of the 8-mer ssDNA lattice. This is represented by the decreasing gradient of chain extension from the 3′ to the 5′-end of the ssDNA oligomer. We suggest that this binding mode is likely to be in dynamic equilibrium with a gp32 monomer conformation (F) in which the C-terminal arm is flipped out of the positive binding cleft and binds to the previously proposed anion binding site (14,15) on the ‘top’ of the gp32 monomer, displacing bound monovalent anions there and permitting full oligonucleotide access to the gp32 binding cleft. Panels (A) and (B) of this figure have been reproduced, with permission, from (12).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: A model for the binding of single gp32 molecules to short ssDNA lattices. (A) Representation of the structure and electrostatic surface potential of the core domain of gp32; blue regions are basic and red regions are acidic (12). (B) The core gp32 domain with a bound ssDNA d(T)4 oligonucleotide fitted to the difference electron density (12). (C) Model of an isolated gp32 molecule showing the C-terminal arm tightly bound to the electropositive cleft of the core domain in the absence of ssDNA. (D) Model of the core gp32 domain bound to an 8-mer ssDNA lattice. The absence of the C-terminal flap allows the oligonucleotide to ‘shuffle’ along the protein lattice. (E) Model of the binding of a gp32 monomer to an 8-mer oligonucleotide. The positively charged binding cleft of the core domain is occluded by the C-terminal arm, thereby hindering the access of the oligonucleotide and resulting in more base unstacking at the 3′ end and less at the 5′ end of the 8-mer ssDNA lattice. This is represented by the decreasing gradient of chain extension from the 3′ to the 5′-end of the ssDNA oligomer. We suggest that this binding mode is likely to be in dynamic equilibrium with a gp32 monomer conformation (F) in which the C-terminal arm is flipped out of the positive binding cleft and binds to the previously proposed anion binding site (14,15) on the ‘top’ of the gp32 monomer, displacing bound monovalent anions there and permitting full oligonucleotide access to the gp32 binding cleft. Panels (A) and (B) of this figure have been reproduced, with permission, from (12).
Mentions: Inspection of the crystal structure of the core DNA-binding domain of gp32 (Figure 6A) suggests that this decrease in access to collisional quenchers near the 3′-end of the 8-mer ssDNA constructs might reflect partial intercalation into the ssDNA chain at these positions of tyrosine side-chains within the DNA binding cleft (see (12)), which could be an additional determinant of the proposed backbone twisting and chain extension resulting from the tight binding of the gp32 to the sugar-phosphate backbones of the ssDNA chain near the 3′-end of the test oligomers.

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