Kinetic Parameters of the Translocation of Bacteriophage T4 Gene 41 Protein Helicase on Single-stranded DNA

Young, Mark C.; Schultz, Danielle M.; Ring, Dawn; Hippel, Peter H. von

doi:10.1006/jmbi.1994.1100

Cited by 74 publications

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“…Further increases in the length of the DNA incrementally push the enzyme into a catalytically more efficient conformation until a maximum is reached at 20 nucleotides or longer. Young et al (29) observed a somewhat similar phenomenon for the gene 41 helicase-primase encoded by bacteriophage T4. The extreme differences in the ability of poly(dT) versus poly(dA) to activate the ATPase activity of the helicase-primase suggests that steric variability in the nucleic acid sequence also affects the manner in which the DNA occupies the binding cleft.…”

Section: Discussionmentioning

confidence: 72%

Interactions of a Subassembly of the Herpes Simplex Virus Type 1 Helicase-Primase with DNA

Healy

You

Dodson

1997

Journal of Biological Chemistry

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The UL5, UL8, and UL52 genes of herpes simplex virus type 1 encode a multisubunit assembly that possesses primase, DNA helicase, and DNA-dependent nucleoside triphosphatase activities. A subassembly consisting of the UL5 and UL52 gene products retains these activities. The nucleoside triphosphatase activity of the UL5/UL52 subassembly is strongly stimulated by both homo-and heteropolymeric single-stranded DNA. Double-stranded DNA has little ability to stimulate the ATPase activity. The subassembly binds both double and single-stranded DNA. Nucleotides are not required for DNA-binding. The minimum length of single-stranded DNA that is bound and that stimulates enzymatic activity is about 12 nucleotides. The kinetic parameters of the ATPase activity of the subassembly are affected by the length of the oligonucleotide coeffector. The K m decreases as the coeffector length is increased up to a length of about 20 nucleotides and then remains independent of coeffector length. The first order rate constant for ATPase activity exhibits a quasihyperbolic dependence on the length of the DNA coeffector and is maximal for coeffectors of 20 nucleotides and longer.Herpes simplex virus type 1 (HSV-1) 1 encodes a heterotrimeric DNA helicase-primase whose subunits are encoded by the UL5, UL8, and UL52 genes of the virus (1, 2). The helicase activity of the enzyme is coupled to the hydrolysis of either ATP or GTP (1, 3). The UL5 gene product possesses a set of domains that correspond with several conserved motifs found in other DNA helicases (4). Mutagenesis of any one of these conserved domains in UL5 protein obliterates viral DNA synthesis (5). The UL52 gene is also essential for viral DNA synthesis and encodes a conserved domain that is found in other primases (6 -8). Mutagenesis of this conserved domain specifically obliterates the primase activity of the HSV-1 helicase-primase (7,8). Deletion of the UL8 gene from HSV-1 renders the virus incapable of DNA replication (9).The HSV-1 helicase-primase can be isolated from insect cells that have been simultaneously infected with recombinant baculoviruses that express each of the three subunits that compose the holoenzyme (10). A subassembly consisting of the UL5 and UL52 gene products also exhibits the DNA-dependent NTPase, DNA helicase, and primase activities that are associated with the holoenzyme (11, 12). The primase activity of this subassembly exhibits DNA sequence dependence and is stimulated by the UL8 protein (13-15). However, purified UL8 protein itself lacks any type of discernible enzymatic activity (11,12).Homologs of the genes encoding the HSV-1 helicase-primase are found in several other human herpesviruses (16). The complex array of activities possessed by the helicase-primases encoded by this group of viruses may be attractive targets for antiviral drug design. An understanding of the properties of the HSV-1 helicase-primase may lead to strategies for developing such compounds.In this work we further examine the interaction of the UL5/ UL52 subassembly with various...

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Section: Discussionmentioning

confidence: 72%

Interactions of a Subassembly of the Herpes Simplex Virus Type 1 Helicase-Primase with DNA

Healy

You

Dodson

1997

Journal of Biological Chemistry

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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%

“…Six subunits of the T4 helicase (gp41) form a hexagon in the presence of ATP or GTP and this helicase translocates 5 0 → 3 0 along the lagging DNA strand template in DNA replication (6), moving in synchrony with both the template-dependent DNA single-nucleotide addition cycle catalyzed by leading and lagging strand T4 DNA polymerases and the NTP binding and hydrolysis cycle that drives the helicase itself (7). The helicase activity of the T4 DNA replication complex resides in a helicase-primase (primosome) complex with a 6∶1 gp41-gp61 subunit ratio (8)(9)(10).…”

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confidence: 99%

“…Fig. 3B shows that DNA duplexes with 2-AP monomer probes at positions 1 and 2 on the leading strand show slight increases in fluorescence intensity with helicase binding (compare red and green bars), whereas the probes in positions 4 and 6 show little or no change ( [4] and [6] constructs). We note that the changes observed at all these positions are small and that the addition of excess GTP (Fig.…”

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confidence: 99%

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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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“…and Dda helicase have been extensively characterized biochemically (2)(3)(4)(5). On the other hand, very little is known about the biochemical properties of UvsW helicase.…”

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confidence: 99%

The T4 Phage UvsW Protein Contains Both DNA Unwinding and Strand Annealing Activities

Nelson¹,

Benkovic

2007

Journal of Biological Chemistry

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UvsW protein belongs to the SF2 helicase family and is one of three helicases found in T4 phage. UvsW governs the transition from origin-dependent to origin-independent replication through the dissociation of R-loops located at the T4 origins of replication. Additionally, in vivo evidence indicates that UvsW plays a role in recombination-dependent replication and/or DNA repair. Here, the biochemical properties of UvsW helicase are described. UvsW is a 3 to 5 helicase that unwinds a wide variety of substrates, including those resembling stalled replication forks and recombination intermediates. UvsW also contains a potent single-strand DNA annealing activity that is enhanced by ATP hydrolysis but does not require it. The annealing activity is inhibited by the non-hydrolysable ATP analog (adenosine 5-O-(thiotriphosphate)), T4 single-stranded DNAbinding protein (gp32), or a small 8.8-kDa polypeptide (UvsW.1). Fluorescence resonance energy transfer experiments indicate that UvsW and UvsW.1 form a complex, suggesting that the UvsW helicase may exist as a heterodimer in vivo. Fusion of UvsW and UvsW.1 results in a 68-kDa protein having nearly identical properties as the UvsW-UvsW.1 complex, indicating that the binding locus of UvsW.1 is close to the C terminus of UvsW. The biochemical properties of UvsW are similar to the RecQ protein family and suggest that the annealing activity of these helicases may also be modulated by protein-protein interactions. The dual activities of UvsW are well suited for the DNA repair pathways described for leading strand lesion bypass and synthesis-dependent strand annealing.

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Kinetic Parameters of the Translocation of Bacteriophage T4 Gene 41 Protein Helicase on Single-stranded DNA

Cited by 74 publications

References 0 publications

Interactions of a Subassembly of the Herpes Simplex Virus Type 1 Helicase-Primase with DNA

Interactions of a Subassembly of the Herpes Simplex Virus Type 1 Helicase-Primase with DNA

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

The T4 Phage UvsW Protein Contains Both DNA Unwinding and Strand Annealing Activities

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