Structure of a repair enzyme interrogating undamaged DNA elucidates recognition of damaged DNA

by Brian Chung

Introduction:
8-oxoguanine (oxoG), produced by spontaneous oxidation of G residues in DNA, is misread as T during replication, giving rise to T-A transversion. The role human 8-oxoguanine DNA glycosylase I (hOGG1) is to recognize and correct oxoG lesion by the base-excision DNA repair pathway. hOGG1 faces a formidable challenge of distinguishing two nucleobases that differ by only two atoms and admitting only oxoG to lesion recognition pocket. A covalent trapping technique was used to determine the structure of a transient complex of hOGG1 and DNA with the normal G base. Comparing the structure of hOGG1-DNA complex presenting oxoG lesion with structure presenting the normal G base shows the mechanism for distinguishing two nucleobases.

Mechanism of hOGG1:
The first step of the gylcosylase activity involves extrusion of damaged base from the genome . This causes local bending of duplex DNA. As the oxoG base is inserted in the lesion recognistion pocket, active site lysine residue (Lys 249) attacks C-1' of the deoxyribose sugar and expels the oxoG base, creating an abasic site. Further enzymatic steps lead to removal of deoxyribose moeity, and incorporation of a correct G nuclotide by DNA polymerase.

Crosslinking of hOGG1 to DNA:
Disulphide trapping implants a disulphide crosslink between protein and DNA to restrict the dissociation. Alkanethiol tether at the N4 position of unpaired C and N149C mutation provides the crosslink for both complexes. Crosslink for complex with oxoG and complex with normal G base .


In addition, K249Q mutation is introduced to the enzyme that binds oxoG containing duplex to produce catalytically inactive mutant, hOGG1-QC . K249 is unmodified when enzyme in crosslink with normal oligonucleotide .

Recognition site:
The most distinct difference is that oxoG is located in the lesion recognition pocket, while G lies against the protein surface at an exo-site outside the pocket.
  • oxoG in recognition site
  • G at exo-site
  • Highlights recognition site

    Interactions between oxoG and amino acids lining the binding pocket
  • Gly 42
  • Gln 315
  • Phe 319 & Cys 253
  • His 270 & Asn 150

    oxoG and G interact very differently with active site residues Phe 319 and His 270

    Energetic basis for discrimination:
    The simulations for binding of oxoG to active site show Lys 249 in the protonated state (-NH3+) and Cys 253 in the deprotonated form (-S-). And quantum mechanical calculations for oxoG and G shows that the primary difference in the electrostatic potentials of the two bases is a charge inversion at positions 7 and 8. This creates local dipoles with opposite directions in oxoG and G. Notably, the dipole moment of Lys249(-NH3+) /Cys253(-S-) pair would be oriented to be antiparallel to oxoG,but parallel to G, if G were to have a similar position as oxoG in the active site.

    Also within the lesion recognition site, an attractive interaction between carbonyl of Gly42 and N7 of oxoG is replaced by a strongly repulsive interaction if G assumed the same position as oxoG in the recognition pocket.

    Conclusions:
    The above results establish that hOGG1 is able to read out the subtle structural distinctions between the oxoG lesion versus its normal counter part G, allowing the lesion admittance to the active-site pocket while rejecting its normal counterpart.

    References:
    1. Banerjee A., Yang W., Karplus M., Verdine G. L. (2005) "Structure of a repair enzyme interrogating undamaged DNA elucidates recognition of damaged DNA", Nature 434, 612-618.

    2. Bruner S. D., Norman D. P. G., Verdine G. L. (2000) "Structural basis for recognition and repair of the endogenous mutagen 8-oxoguanine in DNA", Nature 403, 859-866.