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Methods to study the coupling between replicative helicase and leading-strand DNA polymerase at the replication fork.

ABSTRACT: Replicative helicases work closely with the replicative DNA polymerases to ensure that the genomic DNA is copied in a timely and error free manner. In the replisomes of prokaryotes, mitochondria, and eukaryotes, the helicase and DNA polymerase enzymes are functionally and physically coupled at the leading strand replication fork and rely on each other for optimal DNA strand separation and synthesis activities. In this review, we describe pre-steady state kinetic methods to quantify the base pair unwinding-synthesis rate constant, a fundamental parameter to understand how the helicase and polymerase help each other during leading strand replication. We describe a robust method to measure the chemical step size of the helicase-polymerase complex that determines how the two motors are energetically coupled while tracking along the DNA. The 2-aminopurine fluorescence-based method provide structural information on the leading strand helicase-polymerase complex, such as the distance between the two enzymes, their relative positions at the replication fork, and their roles in fork junction melting. The combined information garnered from these methods informs on the mutual dependencies between the helicase and DNA polymerase enzymes, their stepping mechanism, and their individual functions at the replication fork during leading strand replication.

SUBMITTER: Nandakumar D

PROVIDER: S-EPMC5035585 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Methods to study the coupling between replicative helicase and leading-strand DNA polymerase at the replication fork.

Nandakumar Divya D Patel Smita S SS

Methods (San Diego, Calif.) 20160509

Replicative helicases work closely with the replicative DNA polymerases to ensure that the genomic DNA is copied in a timely and error free manner. In the replisomes of prokaryotes, mitochondria, and eukaryotes, the helicase and DNA polymerase enzymes are functionally and physically coupled at the leading strand replication fork and rely on each other for optimal DNA strand separation and synthesis activities. In this review, we describe pre-steady state kinetic methods to quantify the base pair ...[more]

PMID: 27173619

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Project description:Replicative helicases play central roles in chromosome duplication and their assembly onto DNA is regulated via initiators and helicase loader proteins. The Escherichia coli replicative helicase DnaB and the helicase loader DnaC form a DnaB6-DnaC6 complex that is required for loading DnaB onto single-stranded DNA. Overexpression of dnaC inhibits replication by promoting continual rebinding of DnaC to DnaB and consequent prevention of helicase translocation. Here we show that overexpression of dnaB also inhibits growth and chromosome duplication. This inhibition is countered by co-overexpression of wild-type DnaC but not of a DnaC mutant that cannot interact with DnaB, indicating that a reduction in DnaB6-DnaC6 concentration is responsible for the phenotypes associated with elevated DnaB concentration. Partial defects in the oriC-specific initiator DnaA and in PriA-specific initiation away from oriC during replication repair sensitise cells to dnaB overexpression. Absence of the accessory replicative helicase Rep, resulting in increased replication blockage and thus increased reinitiation away from oriC, also exacerbates DnaB-induced defects. These findings indicate that elevated levels of helicase perturb replication initiation not only at origins of replication but also during fork repair at other sites on the chromosome. Thus, imbalances in levels of the replicative helicase and helicase loader can inhibit replication both via inhibition of DnaB6-DnaC6 complex formation with excess DnaB, as shown here, and promotion of formation of DnaB6-DnaC6 complexes with excess DnaC [Allen GC, Jr., Kornberg A. Fine balance in the regulation of DnaB helicase by DnaC protein in replication in Escherichia coli. J. Biol. Chem. 1991;266:22096-22101; Skarstad K, Wold S. The speed of the Escherichia coli fork in vivo depends on the DnaB:DnaC ratio. Mol. Microbiol. 1995;17:825-831]. Thus, there are two mechanisms by which an imbalance in the replicative helicase and its associated loader protein can inhibit genome duplication.

Dataset Information

Methods to study the coupling between replicative helicase and leading-strand DNA polymerase at the replication fork.

Publications

Methods to study the coupling between replicative helicase and leading-strand DNA polymerase at the replication fork.

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