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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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Related in: MedlinePlus

Suggested mechanism of APEX1 and pol-β interaction.Movement of thumb and 8-kDa subdomains of pol-β changes interface with APEX1 and may trigger complex dissociation. (A) shows 3′ complex APEX1 and pol-β complex with pol-β in open conformation. (B) shows the complex with pol-β in closed conformation. Area of protein-protein interface is circled. Arrows show the direction of thumb and 8-kDa subdomain movement.
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pcbi-1000066-g007: Suggested mechanism of APEX1 and pol-β interaction.Movement of thumb and 8-kDa subdomains of pol-β changes interface with APEX1 and may trigger complex dissociation. (A) shows 3′ complex APEX1 and pol-β complex with pol-β in open conformation. (B) shows the complex with pol-β in closed conformation. Area of protein-protein interface is circled. Arrows show the direction of thumb and 8-kDa subdomain movement.

Mentions: Pol-β binds a cleaved AP site in open conformation [14] inserts a correct nucleotide in closed conformation and returns to the open conformation before it dissociates from the AP site. It is likely that APEX1 performs its 3′-5′ proofreading function for pol-β at this stage [17]. Pol-β then returns to the site to perform the lyase function to remove the dRP residue [18],[19]. We propose the following mechanism for APEX1 and pol-β interaction in the 3′-complex (see Figure 7). After APEX1 has cleaved the AP-site, pol-β in open conformation binds to APEX1, making a single interface comprising segments #2 and #3 and several adjacent residues including those in-between the two segments. The formed complex displaces APEX1 laterally from the cleaved site although both pol-β and APEX1 remain associated with DNA. Transition of pol-β into the closed conformation (precatalytic state) shifts the interface as movement of the 8-kDa domain splits the interface into two distinct segments #2 and #3 and weakens the interaction, while movement of the thumb introduces the new interface segment #1. Once insertion has occurred, the open conformation, still in communication with APEX1, is re-established, allowing a shift for APEX1 3′-exonuclease activity. Pol-β then returns to the site to perform its lyase function.


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)

Suggested mechanism of APEX1 and pol-β interaction.Movement of thumb and 8-kDa subdomains of pol-β changes interface with APEX1 and may trigger complex dissociation. (A) shows 3′ complex APEX1 and pol-β complex with pol-β in open conformation. (B) shows the complex with pol-β in closed conformation. Area of protein-protein interface is circled. Arrows show the direction of thumb and 8-kDa subdomain movement.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1000066-g007: Suggested mechanism of APEX1 and pol-β interaction.Movement of thumb and 8-kDa subdomains of pol-β changes interface with APEX1 and may trigger complex dissociation. (A) shows 3′ complex APEX1 and pol-β complex with pol-β in open conformation. (B) shows the complex with pol-β in closed conformation. Area of protein-protein interface is circled. Arrows show the direction of thumb and 8-kDa subdomain movement.
Mentions: Pol-β binds a cleaved AP site in open conformation [14] inserts a correct nucleotide in closed conformation and returns to the open conformation before it dissociates from the AP site. It is likely that APEX1 performs its 3′-5′ proofreading function for pol-β at this stage [17]. Pol-β then returns to the site to perform the lyase function to remove the dRP residue [18],[19]. We propose the following mechanism for APEX1 and pol-β interaction in the 3′-complex (see Figure 7). After APEX1 has cleaved the AP-site, pol-β in open conformation binds to APEX1, making a single interface comprising segments #2 and #3 and several adjacent residues including those in-between the two segments. The formed complex displaces APEX1 laterally from the cleaved site although both pol-β and APEX1 remain associated with DNA. Transition of pol-β into the closed conformation (precatalytic state) shifts the interface as movement of the 8-kDa domain splits the interface into two distinct segments #2 and #3 and weakens the interaction, while movement of the thumb introduces the new interface segment #1. Once insertion has occurred, the open conformation, still in communication with APEX1, is re-established, allowing a shift for APEX1 3′-exonuclease activity. Pol-β then returns to the site to perform its lyase function.

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