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Base Excision and DNA Binding Activities of Human Alkyladenine DNA Glycosylase

Base Excision and DNA Binding Activities of Human Alkyladenine DNA Glycosylase Are Sensitive to the Base Paired with a Lesion. Abner, C., Lau, A., Ellenberger, T., and Bloom, L. (2001) J. Biol. Chem. 276, 13379-13387. Background. Types of DNA Mutations (spontaneous and/or induced)

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Base Excision and DNA Binding Activities of Human Alkyladenine DNA Glycosylase

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  1. Base Excision and DNA Binding Activities of Human Alkyladenine DNA Glycosylase Are Sensitive to the Base Paired with a Lesion Abner, C., Lau, A., Ellenberger, T., and Bloom, L. (2001) J. Biol. Chem. 276, 13379-13387

  2. Background Types of DNA Mutations (spontaneous and/or induced) - Base pair substitutions (transitions and transversions) - Frameshift mutations (one or two insertions or deletions) - DNA Replication Errors - Base Alterations (tautomerization, deamination, alkylation, and free radical damage of bases) Causes of DNA Mutations - Mutagens (Base analogs, Chemical mutagens, Intercalating agents, and DNA altering structures) - Radiation (Ultraviolet Light, X-rays)

  3. Background DNA Repair Systems - Recognition and repair of mutations of genetic material in DNA. Types of DNA Repair Systems - Damage Reversal (Enzymatic action without backbone disruption) - Damage Removal (Cutting and replacing of damaged bases) - Damage Tolerance (Coping with damage for life continuance) DNA Removal Types - Base Excision Repair (Glycosylases) - Mismatch Repair - Nucleotide Excision Repair

  4. Background Base Excision Repair - Uses Glycosylase enzymes to cut the base-sugar bond. - Different Glycosylase enzymes recognize radiation and chemical DNA damage. - Base recognized and the sugar is cleaved and a new base is inserted by DNA Polymerase. Types of DNA Glycosylases - Uracil Glycosylase (removes uracil from DNA) - Formamidopyrimidine (removes 8-oxoG from DNA) - Human Alkyladenine (removes alkylation-damaged bases)

  5. Purpose Goal To determine whether there are common underlying structural features that are recognized by human alkyladenine DNA glycosylase. Experimentation - examine base excision by hAAG - examine the binding of hAAG to DNA

  6. hAAG Excision Procedure

  7. hAAG Excision Results Abner, C., Lau, A., Ellenberger, T., and Bloom, L. (2001) J. Biol. Chem. 276, 13379-13387

  8. Excision Conclusion Excision - opposing base can affect excision - hydrogen bond donor and hydrogen bond acceptor may play a role in recognition Possible Explanation -enzyme interacts initially with base and base pair or -base-pairing partner affects enzyme interaction with damaged base

  9. Purpose Goal To determine whether there are common underlying structural features that are recognized by human alkyladenine DNA glycosylase. Experimentation - examine base excision by hAAG - examine the binding of hAAG to DNA

  10. hAAG Binding Procedure

  11. hAAG Binding Results Abner, C., Lau, A., Ellenberger, T., and Bloom, L. (2001) J. Biol. Chem. 276, 13379-13387

  12. Binding Conclusion Binding - opposing base can affect binding (In example, Hx affected significantly by opposing base) - hydrogen bond donor and hydrogen bond acceptor may play a role in recognition Possible Explanation - hAAG may not remain bound to DNA after excision

  13. Discussion / Conclusion Important Criteria for Efficient Excision - initial identification of the damaged DNA base - proper alignment of damaged base in enzyme active site Initial Recognition - may depend on recognition of structural distortions in DNA by the damage followed by flipping - may occur solely by flipping damaged base into the enzymatic active site - no fit = no fit for hydrolysis excision

  14. Discussion / Conclusion Base Opposite Damaged Base May Effect Excision - may influence initial recognition of the damage and/or - substrate alignment of damaged base in enzyme active site

  15. Questions ?

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