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An AP endonuclease 1-DNA polymerase beta complex: theoretical prediction of interacting surfaces.

Abyzov A, Uzun A, Strauss PR, Ilyin VA - PLoS Comput. Biol. (2008)

Bottom Line: Analysis of interface behavior during MD simulation and visual inspection of interfaces allowed us to conclude that complexes with pol-beta at the 3'-side of APEX1 are those most likely to occur in vivo.Additional multiple sequence analyses of APEX1 and pol-beta in related organisms identified a set of correlated mutations of specific residues at the predicted interfaces.Based on these results, we propose that pol-beta in the open or closed conformation interacts and makes a stable interface with APEX1 bound to a cleaved abasic site on the 3' side.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Northeastern University, Boston, Massachusetts, United States of America.

ABSTRACT
Abasic (AP) sites in DNA arise through both endogenous and exogenous mechanisms. Since AP sites can prevent replication and transcription, the cell contains systems for their identification and repair. AP endonuclease (APEX1) cleaves the phosphodiester backbone 5' to the AP site. The cleavage, a key step in the base excision repair pathway, is followed by nucleotide insertion and removal of the downstream deoxyribose moiety, performed most often by DNA polymerase beta (pol-beta). While yeast two-hybrid studies and electrophoretic mobility shift assays provide evidence for interaction of APEX1 and pol-beta, the specifics remain obscure. We describe a theoretical study designed to predict detailed interacting surfaces between APEX1 and pol-beta based on published co-crystal structures of each enzyme bound to DNA. Several potentially interacting complexes were identified by sliding the protein molecules along DNA: two with pol-beta located downstream of APEX1 (3' to the damaged site) and three with pol-beta located upstream of APEX1 (5' to the damaged site). Molecular dynamics (MD) simulations, ensuring geometrical complementarity of interfaces, enabled us to predict interacting residues and calculate binding energies, which in two cases were sufficient (approximately -10.0 kcal/mol) to form a stable complex and in one case a weakly interacting complex. Analysis of interface behavior during MD simulation and visual inspection of interfaces allowed us to conclude that complexes with pol-beta at the 3'-side of APEX1 are those most likely to occur in vivo. Additional multiple sequence analyses of APEX1 and pol-beta in related organisms identified a set of correlated mutations of specific residues at the predicted interfaces. Based on these results, we propose that pol-beta in the open or closed conformation interacts and makes a stable interface with APEX1 bound to a cleaved abasic site on the 3' side. The method described here can be used for analysis in any DNA-metabolizing pathway where weak interactions are the principal mode of cross-talk among participants and co-crystal structures of the individual components are available.

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Initial 3′ complex of APEX1 and pol-β (closed conformation).(A) Alignment of DNAs co-crystallized with pol-β and APEX1. X stands for the abasic site and x stands for lesion. Notations c1, c2, and c3 mark the alignments used to produce three initial complexes. (B) View of the 3′ complex structure. APEX1 is on the right and pol-β is on the left. The area of protein-protein interaction is circled.
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pcbi-1000066-g002: Initial 3′ complex of APEX1 and pol-β (closed conformation).(A) Alignment of DNAs co-crystallized with pol-β and APEX1. X stands for the abasic site and x stands for lesion. Notations c1, c2, and c3 mark the alignments used to produce three initial complexes. (B) View of the 3′ complex structure. APEX1 is on the right and pol-β is on the left. The area of protein-protein interaction is circled.

Mentions: For this orientation (see schematic diagram in Figure 1A), initial complexes for pol-β in the closed conformation (PDB-file 2fmq) and APEX1 (PDB-file 1de8) were constructed by aligning the 3′-side of damaged DNA from the APEX1 co-crystal with the 5′-side of the DNA lesion in the pol-β co-crystal. Three complexes termed c1, c2, and c3 satisfied the described requirements (see corresponding alignment in Figure 2A). In the first complex (c1), steric overlaps between APEX1 and pol-β involved more than 10 residues comprising more than 100 atoms in each protein. Polypeptide chains of the two proteins interlaced with each other to produce an unrealistic complex. In the third complex (c3) the interface area was ∼200 Å2 but the ratio of gap volume to area (∼140) was unacceptably large compared to other values in Table 1, indicating very weak interaction, if any. The remaining complex (c2) represented an optimal prediction with only several atoms in steric overlap, which were resolved during MD (see below). The complex is shown in Figure 2B.


An AP endonuclease 1-DNA polymerase beta complex: theoretical prediction of interacting surfaces.

Abyzov A, Uzun A, Strauss PR, Ilyin VA - PLoS Comput. Biol. (2008)

Initial 3′ complex of APEX1 and pol-β (closed conformation).(A) Alignment of DNAs co-crystallized with pol-β and APEX1. X stands for the abasic site and x stands for lesion. Notations c1, c2, and c3 mark the alignments used to produce three initial complexes. (B) View of the 3′ complex structure. APEX1 is on the right and pol-β is on the left. The area of protein-protein interaction is circled.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2289873&req=5

pcbi-1000066-g002: Initial 3′ complex of APEX1 and pol-β (closed conformation).(A) Alignment of DNAs co-crystallized with pol-β and APEX1. X stands for the abasic site and x stands for lesion. Notations c1, c2, and c3 mark the alignments used to produce three initial complexes. (B) View of the 3′ complex structure. APEX1 is on the right and pol-β is on the left. The area of protein-protein interaction is circled.
Mentions: For this orientation (see schematic diagram in Figure 1A), initial complexes for pol-β in the closed conformation (PDB-file 2fmq) and APEX1 (PDB-file 1de8) were constructed by aligning the 3′-side of damaged DNA from the APEX1 co-crystal with the 5′-side of the DNA lesion in the pol-β co-crystal. Three complexes termed c1, c2, and c3 satisfied the described requirements (see corresponding alignment in Figure 2A). In the first complex (c1), steric overlaps between APEX1 and pol-β involved more than 10 residues comprising more than 100 atoms in each protein. Polypeptide chains of the two proteins interlaced with each other to produce an unrealistic complex. In the third complex (c3) the interface area was ∼200 Å2 but the ratio of gap volume to area (∼140) was unacceptably large compared to other values in Table 1, indicating very weak interaction, if any. The remaining complex (c2) represented an optimal prediction with only several atoms in steric overlap, which were resolved during MD (see below). The complex is shown in Figure 2B.

Bottom Line: Analysis of interface behavior during MD simulation and visual inspection of interfaces allowed us to conclude that complexes with pol-beta at the 3'-side of APEX1 are those most likely to occur in vivo.Additional multiple sequence analyses of APEX1 and pol-beta in related organisms identified a set of correlated mutations of specific residues at the predicted interfaces.Based on these results, we propose that pol-beta in the open or closed conformation interacts and makes a stable interface with APEX1 bound to a cleaved abasic site on the 3' side.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Northeastern University, Boston, Massachusetts, United States of America.

ABSTRACT
Abasic (AP) sites in DNA arise through both endogenous and exogenous mechanisms. Since AP sites can prevent replication and transcription, the cell contains systems for their identification and repair. AP endonuclease (APEX1) cleaves the phosphodiester backbone 5' to the AP site. The cleavage, a key step in the base excision repair pathway, is followed by nucleotide insertion and removal of the downstream deoxyribose moiety, performed most often by DNA polymerase beta (pol-beta). While yeast two-hybrid studies and electrophoretic mobility shift assays provide evidence for interaction of APEX1 and pol-beta, the specifics remain obscure. We describe a theoretical study designed to predict detailed interacting surfaces between APEX1 and pol-beta based on published co-crystal structures of each enzyme bound to DNA. Several potentially interacting complexes were identified by sliding the protein molecules along DNA: two with pol-beta located downstream of APEX1 (3' to the damaged site) and three with pol-beta located upstream of APEX1 (5' to the damaged site). Molecular dynamics (MD) simulations, ensuring geometrical complementarity of interfaces, enabled us to predict interacting residues and calculate binding energies, which in two cases were sufficient (approximately -10.0 kcal/mol) to form a stable complex and in one case a weakly interacting complex. Analysis of interface behavior during MD simulation and visual inspection of interfaces allowed us to conclude that complexes with pol-beta at the 3'-side of APEX1 are those most likely to occur in vivo. Additional multiple sequence analyses of APEX1 and pol-beta in related organisms identified a set of correlated mutations of specific residues at the predicted interfaces. Based on these results, we propose that pol-beta in the open or closed conformation interacts and makes a stable interface with APEX1 bound to a cleaved abasic site on the 3' side. The method described here can be used for analysis in any DNA-metabolizing pathway where weak interactions are the principal mode of cross-talk among participants and co-crystal structures of the individual components are available.

Show MeSH
Related in: MedlinePlus