New Biochemical Platforms for the Functional Analysis of DNA Repair Processes

Abstract

Environmental assaults and internal stressors pose continual threats to our genome integrity through the risk of cancer development. Fortunately, our body contains DNA repair proteins to correct cancer-causing mutations on a biologically relevant timescale. Recently, these proteins have been discovered to contain redox-active cofactors such as [4Fe4S] clusters and flavins. The biological roles of these cofactors have been elusive, and some coworkers suggested that these [4Fe4S] motifs might be ancient structural relics. Recent bioinformatics studies on the biogenesis of these redox cofactors hinted otherwise; suggesting that these cofactors might play vital functional roles in living organisms. Our work using a range of biophysical and biochemical techniques showed that the oxidation states of [4Fe4S] clusters control the DNA binding affinities of these bioinorganic repair proteins. By changing the DNA binding affinity, these [4Fe4S] clusters in turn modulate the ability of these proteins to carry out their respective enzymatic actions, such as DNA damage detection. We further developed a biophysical model based on the electrostatic interactions between the [4Fe4S] cluster and DNA to describe the change in DNA binding affinity upon switching the oxidation state of the [4Fe4S] cofactor determined by microscale thermophoresis (MST). Our data also demonstrated that DNA-processing proteins with disparate biological functions but with similar redox potentials can work together to trace and identify DNA lesions. Taken together, our results establish that the redox cofactors regulate the ability of repair proteins to find and fix DNA damage in a timely fashion. Our work delineates a distinct role for these bioinorganic proteins in sensing and repairing DNA damage. The length limit of this redox signaling scheme and its implication in biological magnetosensing will also be discussed.