DNA polymerase κ (Polκ) is involved in a DNA damage tolerance process called translesion synthesis (TLS). TLS is performed by DNA Polκ by accommodating the distortion created by a DNA lesion, thus allowing for successful lesion bypass. The two-step model of lesion bypass involves one polymerase inserting nucleotides across from the lesion and a second DNA polymerase extending past the lesion once a nucleotide has been inserted. Investigation of the lesion bypass ability of DNA Polκ <i>in vitro</i> has shown a limited ability to perform the insertion step and enhanced ability to perform the extension step. In support of Polκ’s ability to perform lesion bypass, the three-dimensional structure of DNA Polκ reveals a constrained active site for the nucleotide insertion step and a more accessible DNA minor-groove region during the extension step. Examination of the Polκ structure uncovers a tyrosine at position 141 that provides stability to the active site structure and a hydrogen bond to the incoming nucleotide. Using site-directed mutagenesis, the tyrosine at position 141 was mutated to phenylalanine (Y141F). The Y141F mutant was overexpressed and purified to homogeneity using recombinant protein expression techniques and affinity chromatography. SDS-PAGE and Bradford analysis were used to determine protein purification and concentration. Using a template-primer extension analysis the role of Y141 was characterized. The Y141F mutant shows a decreased ability to perform DNA synthesis on undamaged DNA; however, the substitution has no effect on the selection of correct vs. incorrect nucleotides.

Tyrosine 141 provides an important hydrogen bond in DNA polymerase κ

255th National Meeting (2018)

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