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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 5′ 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 c4, c5, and c6 mark the alignments used to produce three initial complexes. (B) View of the 5′ complex structure. APEX1 is on the left and pol-β is on the right. The area of protein-protein interaction is circled.
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pcbi-1000066-g004: Initial 5′ 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 c4, c5, and c6 mark the alignments used to produce three initial complexes. (B) View of the 5′ complex structure. APEX1 is on the left and pol-β is on the right. The area of protein-protein interaction is circled.

Mentions: For this orientation (see schematic diagram on Figure 1C), three possible complexes using pol-β in the closed conformation and APEX1 were initially constructed by aligning the 5′-side of the damaged DNA from the APEX1 co-crystal with the 3′-side of the DNA with lesion in the pol-β co-crystal. Three complexes termed c4, c5, and c6 satisfied the described requirements (see corresponding alignment in Figure 4A). In the first complex (c4), steric overlaps of APEX1 and pol-β were large, involving more than 15 residues with more that 150 atoms in each protein. Moreover, polypeptide chains from the two proteins interlaced, producing an unrealistic complex. In the third complex (c6) the proteins hardly touched each other so that the corresponding interface was small with large water filled space between proteins. The remaining complex (c5) represented an optimal prediction with several atoms in steric overlaps, which were resolved during MD (see below). The complex is shown in Figure 4B.


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 5′ 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 c4, c5, and c6 mark the alignments used to produce three initial complexes. (B) View of the 5′ complex structure. APEX1 is on the left and pol-β is on the right. 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-g004: Initial 5′ 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 c4, c5, and c6 mark the alignments used to produce three initial complexes. (B) View of the 5′ complex structure. APEX1 is on the left and pol-β is on the right. The area of protein-protein interaction is circled.
Mentions: For this orientation (see schematic diagram on Figure 1C), three possible complexes using pol-β in the closed conformation and APEX1 were initially constructed by aligning the 5′-side of the damaged DNA from the APEX1 co-crystal with the 3′-side of the DNA with lesion in the pol-β co-crystal. Three complexes termed c4, c5, and c6 satisfied the described requirements (see corresponding alignment in Figure 4A). In the first complex (c4), steric overlaps of APEX1 and pol-β were large, involving more than 15 residues with more that 150 atoms in each protein. Moreover, polypeptide chains from the two proteins interlaced, producing an unrealistic complex. In the third complex (c6) the proteins hardly touched each other so that the corresponding interface was small with large water filled space between proteins. The remaining complex (c5) represented an optimal prediction with several atoms in steric overlaps, which were resolved during MD (see below). The complex is shown in Figure 4B.

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