Single molecule fluorescence studies of replicative helicases

Single Molecule Fluorescence T7 Helicase

Syed, Salman

关键词: Single Molecule Fluorescence T7 Helicase;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/46948/Salman_Syed.pdf?sequence=1&isAllowed=y

美国|英语

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

【 摘 要 】

DNA helicases are motor enzymes that convert the chemical energy of nucleotide triphosphate hydrolysis into mechanical energy for translocation on single stranded (ss) DNA and unwinding of double stranded (ds) DNA.All of the 12 known replicative helicases are hexameric helicases that are ring-shaped in structure and are homohexamers with the exception of the eukaryotic minichromosomal maintenance (MCM) helicase.Their activities are essential for a variety of DNA metabolic transactions including replication, recombination and repair.Bacteriophage T7 gp4 serves as one of the model proteins for replicative helicases.Moreover, the replication machinery of Bacteriophage T7 provides a simple model to study the dynamics of DNA replication.To address how the leading and lagging strand synthesis are coordinated during DNA replication, we employed single molecule Förster Resonance Energy Transfer (smFRET) methods on the Bacteriophage T7 replisome (helicase/primase and polymerase).Our results suggest a model whereby the primase loop allows the coordination of leading and lagging strand DNA synthesis by enabling the newly synthesized RNA primers to remain coupled to the leading strand replication complex and therefore minimizing the delay in lagging strand synthesis.To further investigate the unwinding mechanism of T7 helicase, we used smFRET methods to resolve steps during DNA unwinding by T7 helicase.Our results show direct evidence of larger than 1 bp step size of unwinding by a hexameric helicase.Our studies provide a detailed mechanism of T7 helicase unwinding of double stranded (ds) DNA.Simian Virus (SV) 40 Large T-Antigen (L-tag) serves as a model replicative helicase for studying eukaryotic DNA replication.It belongs to the AAA+ family of helicases that bind to the origin of replication sites on the DNA and form higher order structures to unwind the DNA bidirectionally.We used smFRET methods to probe the unwinding activity of L-tag.An important aspect in understanding the translocation and unwinding mechanisms of ring helicases is how the subunits of these enzymes are coordinated during ATP hydrolysis cycles as they move on ss/dsDNA.E1, another helicase belonging to the AAA+ family of helicases, was shown to utilize a sequential ATP hydrolysis mechanism during ssDNA translocation where individual subunits hydrolyze ATP one at a time.For L-tag, it has been proposed that it utilizes a concerted ATP hydrolysis mechanism where all the subunits hydrolyze ATP simultaneously.To address this question, we performed experiments using L-tag proteins that are deficient in their ATPase and DNA binding activities at different ratios with the wild type L-tag.Our results suggest that individual subunits of the hexamer are coordinated during ATP hydrolysis but do not differentiate between a concerted vs. sequential ATP hydrolysis mechanism.