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Crystal structure of vaccinia virus uracil-DNA glycosylase reveals dimeric assembly.

Schormann N, Grigorian A, Samal A, Krishnan R, DeLucas L, Chattopadhyay D - BMC Struct. Biol. (2007)

Bottom Line: A glycerol molecule is found in the active site of the enzyme in both crystal forms.The new structural features may have evolved for adopting novel functions in the replication machinery of poxviruses.The mode of interaction between the subunits in the dimers suggests a possible model for binding to its partner and the nature of the processivity factor in the polymerase complex.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Biophysical Sciences & Engineering, University of Alabama at Birmingham, Birmingham, AL 35294, USA. nschorm@uab.edu <nschorm@uab.edu>

ABSTRACT

Background: Uracil-DNA glycosylases (UDGs) catalyze excision of uracil from DNA. Vaccinia virus, which is the prototype of poxviruses, encodes a UDG (vvUDG) that is significantly different from the UDGs of other organisms in primary, secondary and tertiary structure and characteristic motifs. It adopted a novel catalysis-independent role in DNA replication that involves interaction with a viral protein, A20, to form the processivity factor. UDG:A20 association is essential for assembling of the processive DNA polymerase complex. The structure of the protein must have provisions for such interactions with A20. This paper provides the first glimpse into the structure of a poxvirus UDG.

Results: Results of dynamic light scattering experiments and native size exclusion chromatography showed that vvUDG is a dimer in solution. The dimeric assembly is also maintained in two crystal forms. The core of vvUDG is reasonably well conserved but the structure contains one additional beta-sheet at each terminus. A glycerol molecule is found in the active site of the enzyme in both crystal forms. Interaction of this glycerol molecule with the protein possibly mimics the enzyme-substrate (uracil) interactions.

Conclusion: The crystal structures reveal several distinctive features of vvUDG. The new structural features may have evolved for adopting novel functions in the replication machinery of poxviruses. The mode of interaction between the subunits in the dimers suggests a possible model for binding to its partner and the nature of the processivity factor in the polymerase complex.

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Model for processivity factor. This figure shows the potential function of vvUDG as a sliding clamp as part of the viral processivity factor and compares the model with the sliding clamp from S. solfataricus. (A) A model of the proposed sliding clamp. Shown is a homotetrameric arrangement of two dimers as observed in both crystal structures of vvUDG. The diameter of the central channel is 27 Å. N-terminal residues 1–16 and C-terminal residues 208–218 that are part of the first dimer interface (see Fig. 2B) and implicated in potential binding to A20 are shown and highlighted (purple). The straight line passing through the central channel indicates a DNA molecule. The possible binding site for A20 is shown. (B) Model of proposed sliding clamp shown as molecular surface colored by electrostatic potential (color code: red electronegative; blue electropositive; white neutral). The view is the same as in Fig. 7A. The electrostatic potential for the highlighted residues in Fig. 7A indicates neutral regions in these locations. (C) Sliding clamp in S. solfataricus (2IX2) [25]. The heterotrimeric sliding clamp PCNA in S. solfataricus is shown as ribbon model (color code: by chain). The diameter of the central channel is 29 Å.
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Figure 7: Model for processivity factor. This figure shows the potential function of vvUDG as a sliding clamp as part of the viral processivity factor and compares the model with the sliding clamp from S. solfataricus. (A) A model of the proposed sliding clamp. Shown is a homotetrameric arrangement of two dimers as observed in both crystal structures of vvUDG. The diameter of the central channel is 27 Å. N-terminal residues 1–16 and C-terminal residues 208–218 that are part of the first dimer interface (see Fig. 2B) and implicated in potential binding to A20 are shown and highlighted (purple). The straight line passing through the central channel indicates a DNA molecule. The possible binding site for A20 is shown. (B) Model of proposed sliding clamp shown as molecular surface colored by electrostatic potential (color code: red electronegative; blue electropositive; white neutral). The view is the same as in Fig. 7A. The electrostatic potential for the highlighted residues in Fig. 7A indicates neutral regions in these locations. (C) Sliding clamp in S. solfataricus (2IX2) [25]. The heterotrimeric sliding clamp PCNA in S. solfataricus is shown as ribbon model (color code: by chain). The diameter of the central channel is 29 Å.

Mentions: A plausible model for the function of poxvirus UDG as part of the processivity factor is shown in Fig. 7. The model shows two dimers arranged around a central channel in a homotetrameric arrangement that is observed in both crystal structures (Figs. 7A and 7B). A similar molecular assembly has been noticed in previously studied sliding clamps [24-26]. The diameter of the central channel in vvUDG is approximately 27 Å (distance measured between corresponding residues on either side). This compares well with diameters in the heterotrimeric sliding clamp of PCNA (Fig. 7C) in Sulfolobus solfataricus (PDBId: 2IX2) and the homodimeric sliding clamp of the polymerase β-subunit in E. coli (PDBId: 1MMI) that are ~29 Å and 30–35 Å, respectively. However, the central channel must have sufficient flexibility in order to accommodate various binding components. In this analogy A20, which acts as a scaffold with binding regions for UDG, E9 and other factors such as D5 and H5 [5], would seem to function as a clamp loader. Protein regions at the type I dimer interface only observed in the trigonal crystal form may be involved in the binding of A20. A part of this dimer interface includes N-terminal residues 1–16 and C-terminal UDG residues 208–218. These 27 residues are almost exclusively hydrophobic. Interestingly, the N-terminal 50 residues of A20 that constitute the minimal interacting binding site for UDG are also predominantly hydrophobic. A hydrophobic interaction in the proposed binding surface is consistent with the observed stability of the heterodimeric A20:UDG complex at high ionic strength (up to 750 mM NaCl) [2]. Although previous experiments have suggested a 1:1 stoichiometry of binding between A20 and UDG [2], the true composition of a functional polymerase unit remains to be determined.


Crystal structure of vaccinia virus uracil-DNA glycosylase reveals dimeric assembly.

Schormann N, Grigorian A, Samal A, Krishnan R, DeLucas L, Chattopadhyay D - BMC Struct. Biol. (2007)

Model for processivity factor. This figure shows the potential function of vvUDG as a sliding clamp as part of the viral processivity factor and compares the model with the sliding clamp from S. solfataricus. (A) A model of the proposed sliding clamp. Shown is a homotetrameric arrangement of two dimers as observed in both crystal structures of vvUDG. The diameter of the central channel is 27 Å. N-terminal residues 1–16 and C-terminal residues 208–218 that are part of the first dimer interface (see Fig. 2B) and implicated in potential binding to A20 are shown and highlighted (purple). The straight line passing through the central channel indicates a DNA molecule. The possible binding site for A20 is shown. (B) Model of proposed sliding clamp shown as molecular surface colored by electrostatic potential (color code: red electronegative; blue electropositive; white neutral). The view is the same as in Fig. 7A. The electrostatic potential for the highlighted residues in Fig. 7A indicates neutral regions in these locations. (C) Sliding clamp in S. solfataricus (2IX2) [25]. The heterotrimeric sliding clamp PCNA in S. solfataricus is shown as ribbon model (color code: by chain). The diameter of the central channel is 29 Å.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 7: Model for processivity factor. This figure shows the potential function of vvUDG as a sliding clamp as part of the viral processivity factor and compares the model with the sliding clamp from S. solfataricus. (A) A model of the proposed sliding clamp. Shown is a homotetrameric arrangement of two dimers as observed in both crystal structures of vvUDG. The diameter of the central channel is 27 Å. N-terminal residues 1–16 and C-terminal residues 208–218 that are part of the first dimer interface (see Fig. 2B) and implicated in potential binding to A20 are shown and highlighted (purple). The straight line passing through the central channel indicates a DNA molecule. The possible binding site for A20 is shown. (B) Model of proposed sliding clamp shown as molecular surface colored by electrostatic potential (color code: red electronegative; blue electropositive; white neutral). The view is the same as in Fig. 7A. The electrostatic potential for the highlighted residues in Fig. 7A indicates neutral regions in these locations. (C) Sliding clamp in S. solfataricus (2IX2) [25]. The heterotrimeric sliding clamp PCNA in S. solfataricus is shown as ribbon model (color code: by chain). The diameter of the central channel is 29 Å.
Mentions: A plausible model for the function of poxvirus UDG as part of the processivity factor is shown in Fig. 7. The model shows two dimers arranged around a central channel in a homotetrameric arrangement that is observed in both crystal structures (Figs. 7A and 7B). A similar molecular assembly has been noticed in previously studied sliding clamps [24-26]. The diameter of the central channel in vvUDG is approximately 27 Å (distance measured between corresponding residues on either side). This compares well with diameters in the heterotrimeric sliding clamp of PCNA (Fig. 7C) in Sulfolobus solfataricus (PDBId: 2IX2) and the homodimeric sliding clamp of the polymerase β-subunit in E. coli (PDBId: 1MMI) that are ~29 Å and 30–35 Å, respectively. However, the central channel must have sufficient flexibility in order to accommodate various binding components. In this analogy A20, which acts as a scaffold with binding regions for UDG, E9 and other factors such as D5 and H5 [5], would seem to function as a clamp loader. Protein regions at the type I dimer interface only observed in the trigonal crystal form may be involved in the binding of A20. A part of this dimer interface includes N-terminal residues 1–16 and C-terminal UDG residues 208–218. These 27 residues are almost exclusively hydrophobic. Interestingly, the N-terminal 50 residues of A20 that constitute the minimal interacting binding site for UDG are also predominantly hydrophobic. A hydrophobic interaction in the proposed binding surface is consistent with the observed stability of the heterodimeric A20:UDG complex at high ionic strength (up to 750 mM NaCl) [2]. Although previous experiments have suggested a 1:1 stoichiometry of binding between A20 and UDG [2], the true composition of a functional polymerase unit remains to be determined.

Bottom Line: A glycerol molecule is found in the active site of the enzyme in both crystal forms.The new structural features may have evolved for adopting novel functions in the replication machinery of poxviruses.The mode of interaction between the subunits in the dimers suggests a possible model for binding to its partner and the nature of the processivity factor in the polymerase complex.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Biophysical Sciences & Engineering, University of Alabama at Birmingham, Birmingham, AL 35294, USA. nschorm@uab.edu <nschorm@uab.edu>

ABSTRACT

Background: Uracil-DNA glycosylases (UDGs) catalyze excision of uracil from DNA. Vaccinia virus, which is the prototype of poxviruses, encodes a UDG (vvUDG) that is significantly different from the UDGs of other organisms in primary, secondary and tertiary structure and characteristic motifs. It adopted a novel catalysis-independent role in DNA replication that involves interaction with a viral protein, A20, to form the processivity factor. UDG:A20 association is essential for assembling of the processive DNA polymerase complex. The structure of the protein must have provisions for such interactions with A20. This paper provides the first glimpse into the structure of a poxvirus UDG.

Results: Results of dynamic light scattering experiments and native size exclusion chromatography showed that vvUDG is a dimer in solution. The dimeric assembly is also maintained in two crystal forms. The core of vvUDG is reasonably well conserved but the structure contains one additional beta-sheet at each terminus. A glycerol molecule is found in the active site of the enzyme in both crystal forms. Interaction of this glycerol molecule with the protein possibly mimics the enzyme-substrate (uracil) interactions.

Conclusion: The crystal structures reveal several distinctive features of vvUDG. The new structural features may have evolved for adopting novel functions in the replication machinery of poxviruses. The mode of interaction between the subunits in the dimers suggests a possible model for binding to its partner and the nature of the processivity factor in the polymerase complex.

Show MeSH
Related in: MedlinePlus