Single Molecule Studies of Polymerase η DNA Interaction with TIRF Microscopy

Abstract

All cellular organisms depend on DNA molecules for the long-term storage of their genetic information. One rather obvious prerequisite for preserving such large amounts of information is a mechanism able to make fast and error proof duplicates despite the presence of damages. The importance of DNA damage repair for a cell's healthy growth becomes evident when considering that there are between 1000 and 1000000 induced DNA lesions in a cell per day.Failure to correct those lesions can lead to serious medical conditions such as Xeroderma Pigmentosum. There for a special class of Polymerases capable of transcribing through such a lesion evolved. One such polymerase is Polymerase η. However, in the case of cancer treatment where such lesions are intentionally induced by chemotherapeutic agents to suppress cancer growth it would be desirable to block the repair mechanism of the low fidelity error prone Polymerase η.There for, to gain insight into the dynamics of the underlying process of lesion-replication, we performed single molecule Forster Resonance Energy Transfer (FRET) experiments with an Total Internal Reflection Fluorescence Microscope (TIRFM). In the assay used, we avoid the difficulties of protein labelling by measuring the induced conformational changes of the template DNA by protein binding. Through these experiments, we where able to obtain information about single molecule binding kinetics of Polymerase η in the presence or absence of nucleotides and also about processivity on different DNA templates. The findings will be contrasted with other high fidelity polymerases such as the Klenow fragment.