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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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Multiple sequence alignment of APEX1 and pol-β.Only alignment for fragments of interacting regions in the 3′ complexes (with open and closed conformation of pol-β) is shown. Residues at the interfaces are in bold; neighboring residues are in normal font. Interacting residues include residues from segments #1, #2, and #3 and adjacent residues (see text), found at interface only in the complex with open conformation of pol-β. Adjacent residues are termed (where possible) AR. Correlated mutations of interacting residues are highlighted in cyan and orange. Other variations in interacting residues are highlighted in red.
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pcbi-1000066-g006: Multiple sequence alignment of APEX1 and pol-β.Only alignment for fragments of interacting regions in the 3′ complexes (with open and closed conformation of pol-β) is shown. Residues at the interfaces are in bold; neighboring residues are in normal font. Interacting residues include residues from segments #1, #2, and #3 and adjacent residues (see text), found at interface only in the complex with open conformation of pol-β. Adjacent residues are termed (where possible) AR. Correlated mutations of interacting residues are highlighted in cyan and orange. Other variations in interacting residues are highlighted in red.

Mentions: The initial complex is shown in Figure 2. Subdomains of pol-β are colored by different colors and named. The protein-protein interface consists of three spatially distinct segments. Residues with correlated mutations (for segment #2: Arg221 of APEX1 and Gln31 of pol-β) are colored in cyan (see also Figure 6).


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)

Multiple sequence alignment of APEX1 and pol-β.Only alignment for fragments of interacting regions in the 3′ complexes (with open and closed conformation of pol-β) is shown. Residues at the interfaces are in bold; neighboring residues are in normal font. Interacting residues include residues from segments #1, #2, and #3 and adjacent residues (see text), found at interface only in the complex with open conformation of pol-β. Adjacent residues are termed (where possible) AR. Correlated mutations of interacting residues are highlighted in cyan and orange. Other variations in interacting residues are highlighted in red.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1000066-g006: Multiple sequence alignment of APEX1 and pol-β.Only alignment for fragments of interacting regions in the 3′ complexes (with open and closed conformation of pol-β) is shown. Residues at the interfaces are in bold; neighboring residues are in normal font. Interacting residues include residues from segments #1, #2, and #3 and adjacent residues (see text), found at interface only in the complex with open conformation of pol-β. Adjacent residues are termed (where possible) AR. Correlated mutations of interacting residues are highlighted in cyan and orange. Other variations in interacting residues are highlighted in red.
Mentions: The initial complex is shown in Figure 2. Subdomains of pol-β are colored by different colors and named. The protein-protein interface consists of three spatially distinct segments. Residues with correlated mutations (for segment #2: Arg221 of APEX1 and Gln31 of pol-β) are colored in cyan (see also Figure 6).

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