Mismatched Base-Pair Simulations for ASFV Pol X/DNA Complexes Help Interpret Frequent G•G Misincorporation

Benedetta A. Sampoli Benítez, Karunesh Arora, Lisa Balistreri, Tamar Schlick

Research output: Contribution to journalArticle

Abstract

DNA polymerase X (pol X) from the African swine fever virus is a 174-amino-acid repair polymerase that likely participates in a viral base excision repair mechanism, characterized by low fidelity. Surprisingly, pol X's insertion rate of the G•G mispair is comparable to that of the four Watson-Crick base pairs. This behavior is in contrast with another X-family polymerase, DNA polymerase β (pol β), which inserts G•G mismatches poorly, and has higher DNA repair fidelity. Using molecular dynamics simulations, we previously provided support for an induced-fit mechanism for pol X in the presence of the correct incoming nucleotide. Here, we perform molecular dynamics simulations of pol X/DNA complexes with different incoming incorrect nucleotides in various orientations [C•C, A•G, and G•G (anti) and A•G and G•G (syn)] and compare the results to available kinetic data and prior modeling. Intriguingly, the simulations reveal that the G•G mispair with the incoming nucleotide in the syn configuration undergoes large-scale conformational changes similar to that observed in the presence of correct base pair (G•C). The base pairing in the G•G mispair is achieved via Hoogsteen hydrogen bonding with an overall geometry that is well poised for catalysis. Simulations for other mismatched base pairs show that an intermediate closed state is achieved for the A•G and G•G mispair with the incoming dGTP in anti conformation, while the protein remains near the open conformation for the C•C and the A•G syn mismatches. In addition, catalytic site geometry and base pairing at the nascent template-incoming nucleotide interaction reveal distortions and misalignments that range from moderate for A•G anti to worst for the C•C complex. These results agree well with kinetic data for pol X and provide a structural/dynamic basis to explain, at atomic level, the fidelity of this polymerase compared with other members of the X family. In particular, the more open and pliant active site of pol X, compared to pol β, allows pol X to accommodate bulkier mismatches such as guanine opposite guanine, while the more structured and organized pol β active site imposes higher discrimination, which results in higher fidelity. The possibility of syn conformers resonates with other low-fidelity enzymes such as Dpo4 (from the Y family), which readily accommodate oxidative lesions.

Original languageEnglish (US)
Pages (from-to)1086-1097
Number of pages12
JournalJournal of Molecular Biology
Volume384
Issue number5
DOIs
StatePublished - Dec 31 2008

Fingerprint

Base Pairing
Nucleotides
DNA
Catalytic Domain
Guanine
Molecular Dynamics Simulation
DNA Repair
African Swine Fever Virus
Protein Conformation
DNA-Directed DNA Polymerase
Hydrogen Bonding
Catalysis
DNA polymerase X
Amino Acids
Enzymes

Keywords

  • ASFV polymerase X
  • induced-fit mechanism
  • mismatch base pair
  • molecular dynamics simulations
  • protein/DNA complex

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Mismatched Base-Pair Simulations for ASFV Pol X/DNA Complexes Help Interpret Frequent G•G Misincorporation. / Sampoli Benítez, Benedetta A.; Arora, Karunesh; Balistreri, Lisa; Schlick, Tamar.

In: Journal of Molecular Biology, Vol. 384, No. 5, 31.12.2008, p. 1086-1097.

Research output: Contribution to journalArticle

Sampoli Benítez, Benedetta A. ; Arora, Karunesh ; Balistreri, Lisa ; Schlick, Tamar. / Mismatched Base-Pair Simulations for ASFV Pol X/DNA Complexes Help Interpret Frequent G•G Misincorporation. In: Journal of Molecular Biology. 2008 ; Vol. 384, No. 5. pp. 1086-1097.
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abstract = "DNA polymerase X (pol X) from the African swine fever virus is a 174-amino-acid repair polymerase that likely participates in a viral base excision repair mechanism, characterized by low fidelity. Surprisingly, pol X's insertion rate of the G•G mispair is comparable to that of the four Watson-Crick base pairs. This behavior is in contrast with another X-family polymerase, DNA polymerase β (pol β), which inserts G•G mismatches poorly, and has higher DNA repair fidelity. Using molecular dynamics simulations, we previously provided support for an induced-fit mechanism for pol X in the presence of the correct incoming nucleotide. Here, we perform molecular dynamics simulations of pol X/DNA complexes with different incoming incorrect nucleotides in various orientations [C•C, A•G, and G•G (anti) and A•G and G•G (syn)] and compare the results to available kinetic data and prior modeling. Intriguingly, the simulations reveal that the G•G mispair with the incoming nucleotide in the syn configuration undergoes large-scale conformational changes similar to that observed in the presence of correct base pair (G•C). The base pairing in the G•G mispair is achieved via Hoogsteen hydrogen bonding with an overall geometry that is well poised for catalysis. Simulations for other mismatched base pairs show that an intermediate closed state is achieved for the A•G and G•G mispair with the incoming dGTP in anti conformation, while the protein remains near the open conformation for the C•C and the A•G syn mismatches. In addition, catalytic site geometry and base pairing at the nascent template-incoming nucleotide interaction reveal distortions and misalignments that range from moderate for A•G anti to worst for the C•C complex. These results agree well with kinetic data for pol X and provide a structural/dynamic basis to explain, at atomic level, the fidelity of this polymerase compared with other members of the X family. In particular, the more open and pliant active site of pol X, compared to pol β, allows pol X to accommodate bulkier mismatches such as guanine opposite guanine, while the more structured and organized pol β active site imposes higher discrimination, which results in higher fidelity. The possibility of syn conformers resonates with other low-fidelity enzymes such as Dpo4 (from the Y family), which readily accommodate oxidative lesions.",
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N2 - DNA polymerase X (pol X) from the African swine fever virus is a 174-amino-acid repair polymerase that likely participates in a viral base excision repair mechanism, characterized by low fidelity. Surprisingly, pol X's insertion rate of the G•G mispair is comparable to that of the four Watson-Crick base pairs. This behavior is in contrast with another X-family polymerase, DNA polymerase β (pol β), which inserts G•G mismatches poorly, and has higher DNA repair fidelity. Using molecular dynamics simulations, we previously provided support for an induced-fit mechanism for pol X in the presence of the correct incoming nucleotide. Here, we perform molecular dynamics simulations of pol X/DNA complexes with different incoming incorrect nucleotides in various orientations [C•C, A•G, and G•G (anti) and A•G and G•G (syn)] and compare the results to available kinetic data and prior modeling. Intriguingly, the simulations reveal that the G•G mispair with the incoming nucleotide in the syn configuration undergoes large-scale conformational changes similar to that observed in the presence of correct base pair (G•C). The base pairing in the G•G mispair is achieved via Hoogsteen hydrogen bonding with an overall geometry that is well poised for catalysis. Simulations for other mismatched base pairs show that an intermediate closed state is achieved for the A•G and G•G mispair with the incoming dGTP in anti conformation, while the protein remains near the open conformation for the C•C and the A•G syn mismatches. In addition, catalytic site geometry and base pairing at the nascent template-incoming nucleotide interaction reveal distortions and misalignments that range from moderate for A•G anti to worst for the C•C complex. These results agree well with kinetic data for pol X and provide a structural/dynamic basis to explain, at atomic level, the fidelity of this polymerase compared with other members of the X family. In particular, the more open and pliant active site of pol X, compared to pol β, allows pol X to accommodate bulkier mismatches such as guanine opposite guanine, while the more structured and organized pol β active site imposes higher discrimination, which results in higher fidelity. The possibility of syn conformers resonates with other low-fidelity enzymes such as Dpo4 (from the Y family), which readily accommodate oxidative lesions.

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