Interactions of Escherichia coli Primary Replicative Helicase DnaB Protein with Single-Stranded DNA. The Nucleic Acid Does Not Wrap around the Protein Hexamer

Bujalowski, Wlodzimierz; Jezewska, Maria J.

doi:10.1021/bi00027a001

Cited by 124 publications

(312 citation statements)

References 31 publications

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“…5, the 5′-end of the ssDNA lagging strand threads through the hole in the center of the gp41 helicase ring and, although the bases do not appear to be constrained, the acrylamide quenching experiments suggest that solvent access to their surfaces is reduced, perhaps as a consequence of the partial shielding of these lagging strand bases from solvent by their position within the ring. The 3′-ssDNA portion of the leading strand, on the other hand, is involved in binding the primosome to the replication fork, likely by interacting with sites on the surfaces of two to three subunits of the hexamer, based on an estimated binding site size of approximately 20 nt (6,15). The presence of the primase subunit significantly tightens the binding interaction of the fork construct with the helicase, although the exact position of the single primase subunit in the initial complex is presently undefined.…”

Section: Discussionmentioning

confidence: 99%

Breathing fluctuations in position-specific DNA base pairs are involved in regulating helicase movement into the replication fork

Jose

Weitzel

Hippel

2012

Proc. Natl. Acad. Sci. U.S.A.

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We previously used changes in the near-UV circular dichroism and fluorescence spectra of DNA base analogue probes placed site specifically to show that the first three base pairs at the fork junction in model replication fork constructs are significantly opened by "breathing" fluctuations under physiological conditions. Here, we use these probes to provide mechanistic snapshots of the initial interactions of the DNA fork with a tight-binding replication helicase in solution. The primosome helicase of bacteriophage T4 was assembled from six (gp41) helicase subunits, one (gp61) primase subunit, and nonhydrolyzable GTPγS. When bound to a DNA replication fork construct this complex advances one base pair into the duplex portion of the fork and forms a stably bound helicase "initiation complex." Replacement of GTPγS with GTP permits the completion of the helicase-driven unwinding process. Our spectroscopic probes show that the primosome in this stable helicase initiation complex binds the DNA of the fork primarily via backbone contacts and holds the first complementary base pair of the fork in an open conformation, whereas the second, third, and fourth base pairs of the duplex show essentially the breathing behavior that previously characterized the first three base pairs of the free fork. These spectral changes, together with dynamic fluorescence quenching results, are consistent with a primosome-binding model in which the lagging DNA strand passes through the central hole of the hexagonal helicase, the leading strand binds to the "outside" surfaces of subunits of the helicase hexamer, and the single primase subunit interacts with both strands.T4 DNA replication complex | helicase mechanisms | 2-aminopurine | DNA structure N ucleic acid bases at single-strand/double-strand (ss-ds) DNA junctions of replication forks and polymerase elongation sites are prime binding targets for proteins and enzymes that manipulate and modify the DNA genome. Determining the conformational changes that occur at these junctions during DNA replication, repair, and transcription can illuminate the mechanisms of these central processes of gene expression. A unique feature of base pairs at and near these ss-dsDNA junctions is that they undergo substantial position-dependent spontaneous opening and closing ("breathing" or "fraying") processes that are driven by thermal fluctuations (1). Proteins that bind more strongly to ss-than to dsDNA sequences can, in principle, use this differential binding free energy to bind these transiently open sequences as a first step in nucleic acid helicase activity, without the immediate expenditure of the chemical free energy of hydrolysis of nucleoside triphosphates (NTPs) (2). Thus, the interactions of genome regulatory proteins with thermally driven DNA breathing events are likely to be important in initiating reactions that involve the exposure and manipulation of single-stranded template sequences at replication forks or transcription bubbles within the normal dsDNA genome. Here, we examine the relations...

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Section: Discussionmentioning

confidence: 99%

Breathing fluctuations in position-specific DNA base pairs are involved in regulating helicase movement into the replication fork

Jose

Weitzel

Hippel

2012

Proc. Natl. Acad. Sci. U.S.A.

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“…RNA sequence 3Ј of the rut site then passes into the hole located at the center of the hexamer. Evidence for passage of a single-stranded nucleic acid substrate through a ring-shaped hexameric structure has been observed for other hexameric helicases, such as DnaB and the T7 gene 4 product (12,13).…”

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confidence: 87%

Transcription Factor Rho Does Not Require a Free End to Act as an RNA-DNA Helicase on an RNA

Burgess

Richardson

2001

Journal of Biological Chemistry

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Escherichia coli Rho factor is a ring-shaped, homohexameric protein that terminates synthesis of RNA through interactions with the nascent RNA transcript. Because its mechanism of action may involve translocation of the RNA transcript through the hole in its ring structure, its action could depend on the availability of a free 5 terminus. To determine whether Rho's activity is 5-end-dependent, its ability to bind to and function on a circular derivative of cro mRNA was investigated. The circular derivative was made in vitro by action of RNA ligase on a derivative of cro RNA containing an extra 10-nucleotide sequence near the 5-end that was complementary to a sequence located near the 3-end. Rho bound nearly as tightly to the circular derivative RNA as to the standard cro transcript. Rho was also able to readily dissociate a DNA oligonucleotide from its helical complex with the circular RNA in an ATP-dependent reaction. Thus, the action of Rho on a transcript does not depend on the availability of a free 5 terminus.Transcription termination factor Rho is an RNA-binding protein that couples the energy derived from ATP hydrolysis to actions that dissociate a RNA transcript from its biosynthetic complex with DNA and RNA polymerase (1, 2). Rho also can dissociate a RNA molecule bound to DNA through a short hybrid helix in a reaction that is dependent on ATP hydrolysis and the presence of an attachment site for Rho on the RNA on the 5Ј side of the hybrid helix (3-5). This RNA-DNA helicase activity may mimic the process of removal of the nascent RNA strand from the transcription elongation complex.Rho factor is believed to function in vivo as a hexamer consisting of six polypeptide subunits arranged in a ring-shaped structure (6, 7). Burgess and Richardson (8) have recently presented a model for the Rho-RNA complex in which a segment of RNA, called a rut site (Rho utilization site), binds to a cleft comprised of the N-terminal RNA-binding domains of the six individual subunits located at one end of the hexameric structure (the crown) (9 -11). RNA sequence 3Ј of the rut site then passes into the hole located at the center of the hexamer. Evidence for passage of a single-stranded nucleic acid substrate through a ring-shaped hexameric structure has been observed for other hexameric helicases, such as DnaB and the T7 gene 4 product (12, 13).The process by which Rho binds to and captures the 3Ј-end of an RNA in the center of the hexamer is not known. Gan and Richardson (14) have recently presented data indicating that Rho, in vitro, could form hexamers by partial assembly of subunits on an RNA. Another mechanism could have the RNA enter into the center of the hexameric structure through an opening or notch in the ring (7). A third mechanism may involve Rho threading onto the nascent transcript from the free 5Ј-end of the RNA. To test the model in which Rho requires a free end of RNA to thread onto in order to function as a terminator, we investigated Rho's ability to utilize its ATP-dependent helicase activity on an RNA lac...

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“…The channel is about 30 Å across and 60 to 80 Å deep (150,176) and could therefore accommodate about 20 bp of double-stranded DNA. It has been estimated independently that the central channel binds a single-stranded DNA fragment 20 Ϯ 3 nucleotides in length (25,79,81,82). The other hexameric helicase whose progress has been reported to be blocked by Tus is SV40 T antigen.…”

Section: Structures Of Dnab and Related Hexameric Helicasesmentioning

confidence: 99%

Replication Termination inEscherichia coli: Structure and Antihelicase Activity of the Tus-TerComplex

Neylon

Kralicek

Hill

et al. 2005

Microbiol Mol Biol Rev

134

144

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The arrest of DNA replication in Escherichia coli is triggered by the encounter of a replisome with a Tus protein-Ter DNA complex. A replication fork can pass through a Tus-Ter complex when traveling in one direction but not the other, and the chromosomal Ter sites are oriented so replication forks can enter, but not exit, the terminus region. The Tus-Ter complex acts by blocking the action of the replicative DnaB helicase, but details of the mechanism are uncertain. One proposed mechanism involves a specific interaction between Tus-Ter and the helicase that prevents further DNA unwinding, while another is that the Tus-Ter complex itself is sufficient to block the helicase in a polar manner, without the need for specific protein-protein interactions. This review integrates three decades of experimental information on the action of the Tus-Ter complex with information available from the Tus-TerA crystal structure. We conclude that while it is possible to explain polar fork arrest by a mechanism involving only the Tus-Ter interaction, there are also strong indications of a role for specific Tus-DnaB interactions. The evidence suggests, therefore, that the termination system is more subtle and complex than may have been assumed. We describe some further experiments and insights that may assist in unraveling the details of this fascinating process

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Interactions of Escherichia coli Primary Replicative Helicase DnaB Protein with Single-Stranded DNA. The Nucleic Acid Does Not Wrap around the Protein Hexamer

Cited by 124 publications

References 31 publications

Breathing fluctuations in position-specific DNA base pairs are involved in regulating helicase movement into the replication fork

Breathing fluctuations in position-specific DNA base pairs are involved in regulating helicase movement into the replication fork

Transcription Factor Rho Does Not Require a Free End to Act as an RNA-DNA Helicase on an RNA

Replication Termination inEscherichia coli: Structure and Antihelicase Activity of the Tus-TerComplex

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