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.
The impact of cationic methylpurines and the effects associated with orienting their methyl groups into the minor or major groove of the DNA duplex are unknown with respect to DNA structure and thermodynamic `signatures'. I propose elucidating these effects by employing stabilized analogues of such lesions, and exploring the relationship between DNA thermodynamic qualities and lesion recognition by DNA repair enzymes.
| Parsons, Zachary D; Bland, Joshua M; Mullins, Elwood A et al. (2016) A Catalytic Role for C-H/? Interactions in Base Excision Repair by Bacillus cereus DNA Glycosylase AlkD. J Am Chem Soc 138:11485-8 |
