PROJECT SUMMARY
Genomic integrity is constantly threatened by ubiquitous alkylating agents found in both endogenous
and exogenous sources. Failure to repair DNA alkylation damage may lead to either or both mutagenesis or
cytotoxicity. DNA is susceptible to alkylation damage, particularly at nucleophilic sites located on purine and
pyrimidine nucleobases. However, these sites are not equivalent, and alkylation of some sites may be more
detrimental to DNA structure, integrity, and to the health of the cell than others. Particularly, alkylation damage
along the major groove of DNA (such as at N7 of guanine and adenine) may affect DNA structure and
dynamics very differently than alkylation along sites located in the minor groove (such as N3 of guanine and
adenine). However, very little information regarding the effects of minor versus major groove lesions exist, in
part due to their inherent instability with respect to spontaneous and enzyme-mediated depurination.
Furthermore, the role of DNA thermodynamic “signatures” in lesion recognition by DNA repair enzymes, such
as DNA glycosylases, is poorly understood, again because such enzymes catalyze the repair of these lesions,
precluding their biochemical/structural study in corresponding protein/DNA complexes.
I propose preparation and incorporation of methylated ribonucleotides 3-methyladenosine (3mA) and 7-
methylguanosine (7mG, in place of their “normal”, deoxyribose counterparts) into duplex DNA, as analogues
with stabilized glycosidic bonds for biophysical, biochemical, and structural studies. Using a combination of
biophysical techniques such as circular dichroism, differential scanning calorimetry, and thermal melting
experiments, I will characterize the effects that methyl groups – a common DNA alkyl lesion – have into the
major and minor groove of duplex DNA (where 7mG and 3mA provide methyl groups located in the major and
minor grooves, respectively). Thermodynamic characteristics of interest include the number of moles of
hydrating water molecules per mole of DNA, the number of moles of associated counterions, B-to-A form DNA
transition propensity (measured in relative humidity upon addition of trifluoroethanol), and DNA duplex melting
temperature. These characteristics give insight regarding how strongly solvated DNA duplexes containing
cationic alkylpurines with minor groove or major groove lesions are, electrostatic/ionic atmosphere changes
associated with these modifications, base stacking/pairing interactions, and general B-form conformational
stability. These properties will then be compared to biochemical data, such as equilibrium binding constants to
DNA glycosylases AAG and AlkD, and will be utilized as structural probes to gain insight regarding recognition
and excision mechanisms employed by these enzymes. Electrophoretic mobility shifts will be used to
determine binding constants, and X-ray crystallography will be used for structural studies.
