Single-Molecule Sorting of Human DNA Repair Enzymes

Abstract

Functional diversity and the dynamic nature of eukaryotic proteome are highly enhanced by posttranslational modifications. Activities of human DNA repair helicases are often controlled by phosphorylation, sumoylation and ubiquitylation. These posttranslational modifications may directly influence the helicase activity or may direct the helicase integration into a particular molecular machine. It is foreseeable that in some cases a few or even a single protein molecule carrying a particular array of modifications could decide the fate of the cell. The presence of a heterogeneous helicase pool in the cell complicates the helicase's biochemical analysis and its structure activity understanding. To this end we have developed single-molecule sorting to quantify, simultaneously probe and distinguish activities of DNA helicases carrying the posttranslational modifications. We surface-tether in vivo biotinylated helicase molecules expressed in human cells. We then use total internal reflection fluorescence microscopy (TIRFM) to follow activity of each individual surface-tethered molecule over multiple cycles of binding and rearrangement of fluorescently labeled DNA substrates or nucleoprotein complexes. The presence of posttranslational modifications is then revealed and is correlated with the measured activity. The difference between the modified and unmodified enzymes can be evaluated even when only a small fraction of helicase molecules carries the modification of interest. Our experimental strategy overcomes difficulties associated with the comprehensive analysis of heterologously modified cellular pool of proteins. It also circumvents another critical hurdle associated with quantitative analysis of large human proteins, namely the difficulty in obtaining large quantities of active protein. I will discuss application of the single-molecule sorting methodology to study two human DNA repair helicases FBH1 and FANCJ.