Chisty, LT; (2014) PcrA function in plasmid replication. Doctoral thesis , UCL (University College London).

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Abstract

PcrA is a DNA helicase involved in unwinding plasmi ds as a part of a complex in asymmetric rolling - circle replication of certain plasmids carrying antibiotic resistance genes. PcrA translocates on single stranded DNA by coupling ATP hydrolysis to movement on DNA. Initiator protein, RepD is required to nick supercoiled plasmid site - specifically and open an ssDNA stretch that PcrA can bind. The presence of RepD is needed throughout plasmid unwinding to maintain processivity. Using fluorescent - based techniques, PcrA helicase mechanistic functions and interactions with different components of the replication complex were examined at the ensemble and single molecule level. The development of ensemble techniques included labelling of PcrA with environment sensitive fluorophores such as the coumarin derivative, MDCC. Using MDCC - PcrA(K138C) a transloc ation assay was developed, which determined that PcrA translocates two times faster on dC (500 bases s -1) than on dT oligos (240 bases s -1). MDCC - PcrA(E449C) was used to investigate the PcrA and RepD interaction. The signal observed with this and other labelled PcrA mutants indicated that the 2B subdomain of PcrA is likely to be interaction site between PcrA and RepD . MDCC - PcrA(E449C) enabled the determination of PcrA kin etics with the initiation complex and showed that ATP - PcrA binds differently to PcrA to RepD - DNA complex as compared to apo PcrA , possibly indicating tha t ATP binding stabilises PcrA in a specific conformation when binding to RepD - DNA. A single - molecule a ssay was developed to study individual surface - bound PcrA helicases unwinding full length plasmids by total internal reflection fluorescence microscop y . PcrA unwinds plasmid with the average rate of 40 - 50 bp s - 1 showing 10 - fold variation between the indivi dual helicases. The experiments indicated that the unwinding starts almost immediately after addition of ATP . The single molecule experiments with fluorophore - labelled biotinylated PcrA so clear indication that PcrA unwinds plasmids as a monomer with proce ssivity over 3000 base pairs. The comparison of the ssDNA translocation rate and dsDN A unwinding rates show ed a significant difference between the two forms of translocation, which indicate d PcrA to be a passive helicase. This means that PcrA is n o t active ly destabilising the hydrogen bonding between DNA bases but taking advantage of the thermal fluctuation between the base pairs and so leading to separation of component DNA strands.

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