Carcinogens are substances which are capable of chemically modifying DNA via covalent bonds, altering the coding potential of affected DNA. A reported major constituent of cigarette smoke, the enzymatically activated compound (+)benzo[a]pyrene-7,8-dihyrdodiol-9,10-epoxide (BPDE) is listed as a Group 1 carcinogen by the International Agency for Research on Cancer i.e. BDPE is highly carcinogenic to humans and also poses a high exposure risk. DNA adducted with BPDE (N2-BPDEdG) cannot be replicated by classical DNA polymerases however, human DNA polymerase kappa (pol?) has been shown to perform replication across similar bulky N2-dG adducts efficiently; resisting a tendency to create frameshift mutations. Little information is known about the mechanism by which pol? is able to perform DNA synthesis across N2-BPDEdG adducts but data suggests that the novel domain of pol?, termed the N-clasp, which has been shown as essential for replicative function of undamaged DNA is vital in this process. To better understand the role of pol? in the bypass of BPDE adducts single residue variants were produced and screened for functionality. A total of ten catalectic core (aa68-526) residues F49, D107, R144, P169, F192, E199, E292, R298, K321 were selected for mutagenic screening using a single nucleotide polymorphism (SNP) database, computational methods THEMATICS and POOL, and computational structural analysis. Screening of the variants includes primer extension assays to measure nucleotide incorporation and fluorescence anisotropy assays to measure DNA-Enzyme complex binding strengths.

Mutagensis and functionalty screening of the catylctic core of human DNA pol?

247th National Meeting (2014)

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