Base Excision and DNA Binding Activities

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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
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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)
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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
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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)
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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
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hAAG Excision Procedure
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hAAG Excision Results
Abner, C., Lau, A., Ellenberger, T., and Bloom,
L. (2001) J. Biol. Chem. 276, 13379-13387
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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
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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
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hAAG Binding Procedure
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hAAG Binding Results
Abner, C., Lau, A., Ellenberger, T., and Bloom,
L. (2001) J. Biol. Chem. 276, 13379-13387
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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 Possib
le Explanation - hAAG may not remain bound to DNA
after excision
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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
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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
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Questions ?