Mutations in the genetic code are often the result of error prone replication past damage to the nucleotides that comprise the sequence.  DNA polymerase kappa (Polk), a member of the Y-family, has been shown to possess the ability of allowing replication to proceed past a site containing damage from carcinogens as well as chemotherapeutic agents.  The goal of this project was to develop an assay utilizing Fluorescence Resonance Energy Transfer (F.R.E.T.) as a means of analyzing the conformational dynamics of human DNA polymerase kappa (Polk) as it performs lesion bypass.  A truncated version of Pol kappa was used containing amino acids 19 through 526(Polk19-526), which has been shown to contain the same catalytic functionality as the full length protein.  Alexa Fluor-488 c-5 Maleimide dye was the probe chosen for this project as it reacts with the sulfur group of Cysteine, of which there are only 5 present within the truncated protein.  A DTNB assay was used to determine which of the 5 Cysteine residues would be accessible to the Alexa probe.  I will present my results of the DTNB assays and discuss our current progress into the conformational dynamics of Polk during DNA lesion bypass.

Mutation of human DNA polymerase kappa to a single cysteine variant for the purpose of developing a molecular tool used in fluorescence based assays

247th National Meeting (2014)

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