Single-molecule study of the DNA helicase regulating genomic stability

Single molecule;Föster resonance energy transfer (FRET);Replication;Recombination;Helicase;PcrA;UvrD;RecA;XPD

Park, Jeehae

关键词: Single molecule;    Föster resonance energy transfer (FRET);    Replication;    Recombination;    Helicase;    PcrA;    UvrD;    RecA;    XPD;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/29546/Park_Jeehae.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

A DNA helicase, PcrA is an essential protein in gram-positive bacteria. PcrA utilizes its helicase activity and translocation activity to regulate various cellular functions including plasmid replication and counteracting deleterious recombination. Using single molecule FRET technique and site-directed mutation, we discovered enzymes new activity: PcrA act as a powerful motor and remove RecA protein effectively from single strand DNA (ssDNA) as it pulls the RecA-coated lagging strand from the DNA junction. Our observation provides plausible mechanistic explanation for how a helicase can protect stalled replication fork from uncontrolled recombination in vivo. PcrA is a prototypical translocating motor of which structure and kinetics are heavily studied for its kinetic mechanism. Utilizing the persistent reeling-in activity of PcrA, we conducted heterogeneity-free analysis and identified that the fundamental unit of translocation kinetics is a ‘one nucleotide uniform step’. Our result reconciles debates between the structural and biochemical studies on the motor mechanism, and moreover, clarifies a popular oversight regarding molecular heterogeneity.Next, I developed new detection scheme to monitor translocation dynamics of unlabeled protein that carries metal containing domain. By utilizing protein’s ability to quench organic fluorophore in distance dependent manner, XPD helicase translocation on ssDNA was monitored and, the differential effect of single-strand binding protein, RPAs on the XPD translocation kinetics were evaluated.Lastly, I aim to observe translocating motor on a physiological length of ssDNA. FRET has limited working distance and therefore about 80 nucleotides (nt) or further distance change is undetectable. Custom made DNA nano-structure was devised and it is capable of providing up to 2000 nt stretched ssDNA tracks immobilized on the surface. I use fluorescence colocalization and FIONA technique to monitor hundreds of nanometer scale motion of translocases and activity of other single strand interacting proteins.