Identification and Purification of a Protein that Stimulates the Helicase Activity of the Escherichia coli Rep Protein

Escherichia coli UvrD DNA helicase functions in several DNA repair processes. As a monomer, UvrD can translocate rapidly and processively along ssDNA; however, the monomer is a poor helicase. To unwind duplex DNA in vitro, UvrD needs to be activated either by self-assembly to form a dimer or by interaction with an accessory protein. However, the mechanism of activation is not understood. UvrD can exist in multiple conformations associated with the rotational conformational state of its 2B subdomain, and its helicase activity has been correlated with a closed 2B conformation. Using single-molecule total internal reflection fluorescence microscopy, we examined the rotational conformational states of the 2B subdomain of fluorescently labeled UvrD and their rates of interconversion. We find that the 2B subdomain of the UvrD monomer can rotate between an open and closed conformation as well as two highly populated intermediate states. The binding of a DNA substrate shifts the 2B conformation of a labeled UvrD monomer to a more open state that shows no helicase activity. The binding of a second unlabeled UvrD shifts the 2B conformation of the labeled UvrD to a more closed state resulting in activation of helicase activity. Binding of a monomer of the structurally similar Escherichia coli Rep helicase does not elicit this effect. This indicates that the helicase activity of a UvrD dimer is promoted via direct interactions between UvrD subunits that affect the rotational conformational state of its 2B subdomain.

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The Escherichia coli rep mutation. X. Consequences of increased and decreased Rep protein levels

MGG Molecular & General Genetics ◽

10.1007/bf00329669 ◽

1987 ◽

Vol 209 (2) ◽

pp. 382-390 ◽

Cited By ~ 38

Author(s):

Joseph Colasanti ◽

David T. Denhardt

Keyword(s):

Escherichia Coli ◽

Rep Protein ◽

Protein Levels

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Structure and function of Escherichia coli DnaB protein: Role of the N-terminal domain in helicase activity

Biochemistry ◽

10.1021/bi00203a028 ◽

1994 ◽

Vol 33 (37) ◽

pp. 11307-11314 ◽

Cited By ~ 28

Author(s):

Subhasis B. Biswas ◽

Pei-Hua Chen ◽

Esther E. Biswas

Keyword(s):

Escherichia Coli ◽

Structure And Function ◽

Helicase Activity ◽

Terminal Domain ◽

And Function

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Enzyme-catalyzed DNA unwinding: Studies on Escherichia coli rep protein

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.76.4.1658 ◽

1979 ◽

Vol 76 (4) ◽

pp. 1658-1662 ◽

Cited By ~ 141

Author(s):

G. T. Yarranton ◽

M. L. Gefter

Keyword(s):

Escherichia Coli ◽

Dna Unwinding ◽

Rep Protein

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Mechanism and Control of Interspecies Recombination in Escherichia coli. I. Mismatch Repair, Methylation, Recombination and Replication Functions

Genetics ◽

10.1093/genetics/150.2.533 ◽

1998 ◽

Vol 150 (2) ◽

pp. 533-542 ◽

Cited By ~ 4

Author(s):

Snježana Štambuk ◽

Miroslav Radman

Keyword(s):

Escherichia Coli ◽

Mismatch Repair ◽

Dna Sequences ◽

Sequence Divergence ◽

Repair Process ◽

Helicase Activity ◽

Dam Methylation ◽

Homeologous Recombination ◽

And Control ◽

Interspecies Recombination

Abstract A genetic analysis of interspecies recombination in Escherichia coli between the linear Hfr DNA from Salmonella typhimurium and the circular recipient chromosome reveals some fundamental aspects of recombination between related DNA sequences. The MutS and MutL mismatch binding proteins edit (prevent) homeologous recombination between these 16% diverged genomes by at least two distinct mechanisms. One is MutH independent and presumably acts by aborting the initiated recombination through the UvrD helicase activity. The RecBCD nuclease might contribute to this editing step, presumably by preventing reiterated initiations of recombination at a given locus. The other editing mechanism is MutH dependent, requires unmethylated GATC sequences, and probably corresponds to an incomplete long-patch mismatch repair process that does not depend on UvrD helicase activity. Insignificant effects of the Dam methylation of parental DNAs suggest that unmethylated GATC sequences involved in the MutH-dependent editing are newly synthesized in the course of recombination. This hypothetical, recombination-associated DNA synthesis involves PriA and RecF functions, which, therefore, determine the extent of MutH effect on interspecies recombination. Sequence divergence of recombining DNAs appears to limit the frequency, length, and stability of early heteroduplex intermediates, which can be stabilized, and the recombinants mature via the initiation of DNA replication.

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