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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

The interface of APEX1 and pol-β (closed conformation) in 3′ complex after 0.2 ns of MD simulation.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).
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pcbi-1000066-g003: The interface of APEX1 and pol-β (closed conformation) in 3′ complex after 0.2 ns of MD simulation.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).

Mentions: Analysis of the complex allowed identification of potential interacting residues of APEX1 and pol-β (see Table 2). The interface of APEX1 contained 16 residues with six, Arg221, Asn222, Lys224, Gln235, Ser275 and Lys276, representing the largest interface surface. The interface of pol-β contained 13 residues with seven, Gln31, Ile33, His34, Ser109, Lys113, Gly305 and Val306, contributing the largest interface area. Overall the interface consisted of three distinct spatial segments (Figure 3). In pol-β the interfaces of each segment were composed of residues from different subdomains: in segment #1 from the thumb subdomain, in segment #2 from the 8-kD subdomain and in segment #3 from the finger subdomain. Segments #1 and #3 were smaller then segment #2. The segments behave differently during the simulation (see Table 1). The areas of segments #2 and #3 were essentially stable while the area of segment #1 fluctuated (see Table 2). Not all of the amino acid residues in the segment #1 participated in interaction at all times.


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)

The interface of APEX1 and pol-β (closed conformation) in 3′ complex after 0.2 ns of MD simulation.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).
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1000066-g003: The interface of APEX1 and pol-β (closed conformation) in 3′ complex after 0.2 ns of MD simulation.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).
Mentions: Analysis of the complex allowed identification of potential interacting residues of APEX1 and pol-β (see Table 2). The interface of APEX1 contained 16 residues with six, Arg221, Asn222, Lys224, Gln235, Ser275 and Lys276, representing the largest interface surface. The interface of pol-β contained 13 residues with seven, Gln31, Ile33, His34, Ser109, Lys113, Gly305 and Val306, contributing the largest interface area. Overall the interface consisted of three distinct spatial segments (Figure 3). In pol-β the interfaces of each segment were composed of residues from different subdomains: in segment #1 from the thumb subdomain, in segment #2 from the 8-kD subdomain and in segment #3 from the finger subdomain. Segments #1 and #3 were smaller then segment #2. The segments behave differently during the simulation (see Table 1). The areas of segments #2 and #3 were essentially stable while the area of segment #1 fluctuated (see Table 2). Not all of the amino acid residues in the segment #1 participated in interaction at all times.

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