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The regulation mechanism of the C-terminus of RecA proteins during DNA strand-exchange process.

ABSTRACT: The C-terminus of Escherichia coli RecA protein can affect the DNA binding affinity, interact with accessory proteins, and regulate the RecA activity. A substantial upward shift in the pH-reaction profile of RecA-mediated DNA strand-exchange reactions was observed for C-terminal-truncated E. coli ΔC17 RecA, Deinococcus radiodurans RecA, and Deinococcus ficus RecA. Here, the process of RecA-mediated strand exchange from the beginning to the end was investigated with florescence resonance energy transfer and tethered particle motion experiments to determine the detailed regulation mechanism. RecA proteins with a shorter C-terminus possess more stable nuclei, higher DNA binding affinities, and lower protonation requirements for the formation of nucleoprotein filaments. Moreover, more stable synaptic complexes in the homologous sequence searching process were also observed for RecA proteins with a shorter C-terminus. Our results suggest that the C-terminus of RecA proteins regulates not only the formation of RecA nucleoprotein filaments but also the entrance of secondary DNA into RecA nucleoprotein filaments.

SUBMITTER: Fan HF

PROVIDER: S-EPMC8390967 | biostudies-literature | 2021 Aug

REPOSITORIES: biostudies-literature

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The regulation mechanism of the C-terminus of RecA proteins during DNA strand-exchange process.

Fan Hsiu-Fang HF Su Shu S

Biophysical journal 20210629 15

The C-terminus of Escherichia coli RecA protein can affect the DNA binding affinity, interact with accessory proteins, and regulate the RecA activity. A substantial upward shift in the pH-reaction profile of RecA-mediated DNA strand-exchange reactions was observed for C-terminal-truncated E. coli ΔC17 RecA, Deinococcus radiodurans RecA, and Deinococcus ficus RecA. Here, the process of RecA-mediated strand exchange from the beginning to the end was investigated with florescence resonance energy t ...[more]

PMID: 34197804

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Project description:The type I DNA topoisomerase from vaccinia virus (vTopo) forms a reversible covalent 3'-phosphotyrosyl linkage with a single strand of duplex DNA at the preferred sequence 5'-(C/T)CCTTp downward arrowN(-1)N(-2)N(-3)-3'. The enzyme-DNA covalent adduct is recombinogenic in cells, because the nicked strand downstream of the cleavage site can dissociate and be replaced by another DNA strand, potentially resulting in genome rearrangements if the enzyme executes strand ligation. Topo I could play an active role in strand exchange, either by altering the kinetics or thermodynamics of DNA strand binding or by serving as a proofreading gate to prevent ligation of incoming DNA strands containing mismatches. To address these questions, we have measured the kinetic and thermodynamic parameters for strand annealing to a purified vaccinia Topo I-DNA (vTopo-DNA) covalent complex containing a single-strand overhang and then compared them with the same overhang duplex in the absence of vTopo. We found that vTopo accelerates the strand association rate by 2-fold but has no effect on the rate of strand dissociation. vTopo has a similar small effect on the annealing parameters of a series of DNA strands containing single mismatches. In contrast, single base mismatches at the -1, -2, or -3 positions decreased the forward rate and equilibrium constant for reversible strand ligation by 10-fold. These data establish that while vTopo is a bystander during the annealing step of strand exchange, the enzyme strongly discriminates against mismatches close to the cleavage site during the subsequent events leading to strand ligation. A mechanism emerges where vTopo oscillates between an open state where the downstream DNA segment does not interact with the enzyme and a closed state where catalytically important contacts are formed with this region. This oscillation between an open and closed state of the covalently bound enzyme is likely important for regulating the number of DNA superhelical turns that are removed during the lifetime of the covalent complex with supercoiled substrates.

Dataset Information

The regulation mechanism of the C-terminus of RecA proteins during DNA strand-exchange process.

Publications

The regulation mechanism of the C-terminus of RecA proteins during DNA strand-exchange process.

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