Single Molecule Study on Incorporation Efficiency of DPO4 and Klenow Fragment in the Presence of BPDE Adduct

Abstract

It's well known that the binding of DNA adducts such as benzo[a]pyrene-diol-epoxide (BPDE) to DNA template strand can impede or block DNA synthesis during the process of DNA replication. While DNA synthesis involving high fidelity replicative A-family polymerases such as Klenow fragment are blocked by DNA adducts, members of Y-family DNA polymerases such as Sulfolobus solfataricus P2 DNA polymerase IV (DPO4) can bypass the DNA adducts and resume the DNA synthesis. Understanding the functional differences between A-family and Y-family DNA polymerases in the process of DNA replication and the mechanism of bypassing DNA adducts is of great value to explain the cause of mutagenesis. We introduce an assay by anchoring DNA molecules to the modified surface to study the incorporation efficiency of DPO4 and Klenow fragment with the presence of BPDE adduct at single molecule level. Specifically, we anchor fluorescent labeled DNA template onto this surface with the adduct site open for nucleotide incorporation, photobleach the labels and flow the polymerases and labeled nucleotides into the hybridization cell. Using Total Internal Reflection Fluorescence Microscopy (TIRFM) we identify the time sequence incorporation of the nucleotides onto the anchored DNA template by identifying the location of the labeled nucleotide from TIRF images. We further quantify the signal densities of the images obtained from the two different polymerases, thus examining whether incorporation reactions have been executed and quantifying the incorporation efficiency of the polymerases.