Role of active site tyrosines in dynamic aspects of DNA binding by AP endonuclease☆

Melo, Luisa F.; Mundle, Sophia T.; Fattal, Michael H.; O’Regan, N. Edel

doi:10.1016/j.dnarep.2006.11.004

Cited by 7 publications

(8 citation statements)

References 47 publications

(68 reference statements)

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“…20,22,24,25,[27][28][29][30][31][32][33][34][35][36][37][38][39][40] hApe1 was shown to orient the AP site containing DNA via positively charged complementary surfaces and to insert loops into the helical base stack. The resulting DNA bent and kink causes the AP site to flip out into an extra-helical conformation within the active-site pocket.…”

Section: Introductionmentioning

confidence: 99%

Crystal Structure Analysis of DNA Uridine Endonuclease Mth212 Bound to DNA

Lakomek

Dickmanns

Ciirdaeva

et al. 2010

Journal of Molecular Biology

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Section: Introductionmentioning

confidence: 99%

Crystal Structure Analysis of DNA Uridine Endonuclease Mth212 Bound to DNA

Lakomek

Dickmanns

Ciirdaeva

et al. 2010

Journal of Molecular Biology

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“…Furthermore, in several recent publications, the importance of conserved Tyr 171 for AP site recognition, binding, and catalysis was reported (38–40). The substitution of a Phe residue for Tyr 171 significantly decreases the catalytic efficiency of APE1.…”

mentioning

confidence: 96%

Conformational Transitions in Human AP Endonuclease 1 and Its Active Site Mutant during Abasic Site Repair

et al. 2010

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AP endonuclease 1 (APE 1) is a crucial enzyme of the base excision repair pathway (BER) in human cells. APE1 recognizes apurinic/apyrimidinic (AP) sites and makes a nick in the phosphodiester backbone 5′ to them. The conformational dynamics and presteady-state kinetics of wild-type APE1 and its active site mutant, Y171F-P173L-N174K, have been studied. To observe conformational transitions occurring in the APE1 molecule during the catalytic cycle, we detected intrinsic tryptophan fluorescence of the enzyme under single turnover conditions. DNA duplexes containing a natural AP site, its tetrahydrofuran analogue, or a 2′-deoxyguanosine residue in the same position were used as specific substrates or ligands. The stopped-flow experiments have revealed high flexibility of the APE1 molecule and the complexity of the catalytic process. The fluorescent traces indicate that wild-type APE1 undergoes at least four conformational transitions during the processing of abasic sites in DNA. In contrast, nonspecific interactions of APE1 with undamaged DNA can be described by a two-step kinetic scheme. Rate and equilibrium constants were extracted from the stopped-flow and fluorescence titration data for all substrates, ligands, and products. A replacement of three residues at the enzymatic active site including the replacement of tyrosine 171 with phenylalanine in the enzyme active site resulted in a 2 × 104-fold decrease in the reaction rate and reduced binding affinity. Our data indicate the important role of conformational changes in APE1 for substrate recognition and catalysis.

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“…Since AP sites are pre-mutagenic lesions that can prevent normal DNA replication and transcription, the cell contains systems to identify and repair such sites, specifically the base excision repair (BER) pathway [1] . Apurinic/apyrimidinic endonuclease 1 (APEX1) cleaves the phosphodiester backbone 5′ to the AP site [2] – [4] . The cleavage, which is a key step in the BER pathway, is followed by nucleotide insertion and removal of the downstream deoxyribose moiety, performed most often by DNA polymerase beta (pol-β) [5] .…”

Section: Introductionmentioning

confidence: 99%

An AP Endonuclease 1–DNA Polymerase β Complex: Theoretical Prediction of Interacting Surfaces

2008

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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-β). While yeast two-hybrid studies and electrophoretic mobility shift assays provide evidence for interaction of APEX1 and pol-β, the specifics remain obscure. We describe a theoretical study designed to predict detailed interacting surfaces between APEX1 and pol-β 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-β located downstream of APEX1 (3′ to the damaged site) and three with pol-β 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 (∼−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-β at the 3′-side of APEX1 are those most likely to occur in vivo. Additional multiple sequence analyses of APEX1 and pol-β in related organisms identified a set of correlated mutations of specific residues at the predicted interfaces. Based on these results, we propose that pol-β 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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Role of active site tyrosines in dynamic aspects of DNA binding by AP endonuclease☆

Cited by 7 publications

References 47 publications

Crystal Structure Analysis of DNA Uridine Endonuclease Mth212 Bound to DNA

Crystal Structure Analysis of DNA Uridine Endonuclease Mth212 Bound to DNA

Conformational Transitions in Human AP Endonuclease 1 and Its Active Site Mutant during Abasic Site Repair

An AP Endonuclease 1–DNA Polymerase β Complex: Theoretical Prediction of Interacting Surfaces

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