Domain Effects on the DNA-interactive Properties of Bacteriophage T4 Gene 32 Protein

Waidner, Lisa A.; Flynn, Elizabeth K.; Wu, Min; Li, Xing; Karpel, Richard L.

doi:10.1074/jbc.m007778200

Cited by 26 publications

(47 citation statements)

References 25 publications

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“…[25][26][27][28][29] Our results are consistent with intramolecular association of the C-terminal domain to the core domain in pre-equilibrium to the binding of gp32 to both dsDNA and ssDNA 8 . As the salt concentration is lowered, the expected increase in protein-DNA binding affinity is counteracted by the stronger binding of the acidic flap to the core domain, effectively regulating the protein's capability to destabilize DNA 8; 9; 30 .…”

Section: Introductionsupporting

confidence: 76%

Modulation of T4 gene 32 protein DNA binding activity by the recombination mediator protein UvsY

Pant

Shokri

Karpel

et al. 2008

Journal of Molecular Biology

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Bacteriophage T4 UvsY is a recombination mediator protein that promotes assembly of the UvsXssDNA presynaptic filament. UvsY helps UvsX to displace T4 gene 32 protein (gp32) from ssDNA, a reaction necessary for proper formation of the presynaptic filament. Here we use DNA stretching to examine UvsY interactions with single DNA molecules in the presence and absence of gp32 and a gp32 C-terminal truncation (*I), and show that in both cases UvsY is able to destabilize gp32-ssDNA interactions. In these experiments UvsY binds more strongly to dsDNA than ssDNA due to its inability to wrap ssDNA at high forces. To support this hypothesis, we show that ssDNA created by exposure of stretched DNA to glyoxal is strongly wrapped by UvsY, but wrapping occurs only at low forces. Our results demonstrate that UvsY interacts strongly with stretched DNA in the absence of other proteins. In the presence of gp32 and *I, UvsY is capable of strongly destabilizing gp32-DNA complexes in order to facilitate ssDNA wrapping, which in turn prepares the ssDNA for presynaptic filament assembly in the presence of UvsX. Thus, UvsY mediates UvsX binding to ssDNA by converting rigid gp32-DNA filaments into a structure that can be strongly bound by UvsX.

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Section: Introductionsupporting

confidence: 76%

Modulation of T4 gene 32 protein DNA binding activity by the recombination mediator protein UvsY

Pant

Shokri

Karpel

et al. 2008

Journal of Molecular Biology

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“…Previous results showed strong salt-dependent binding for gp32 and its fragments, although bulk experiments could only be performed in high salt (>300 mM Na + , typically). 96,98 Under these conditions, *I was observed to bind somewhat more strongly to ssDNA than gp32. 96 Both proteins were observed to bind with a cooperativity parameter !…”

Section: Figurementioning

confidence: 95%

“…Furthermore, extrapolation from high salt conditions prompted the expectation that both gp32 and *I should lower the melting temperature of dsDNA by $508C, although subsequent experiments confirmed a change in the melting temperature for *I only. 96,98,99 Optical tweezers stretching data are shown for extension (solid points) and relaxation (open points) cycles in Figure 9. 100 Data are shown for DNA without protein, in the presence of 200 nM gp32 and 200 nM *I.…”

Section: Figurementioning

confidence: 99%

Mechanisms of DNA binding determined in optical tweezers experiments

McCauley¹,

Williams²

2006

Biopolymers

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The last decade has seen rapid development in single molecule manipulation of RNA and DNA. Measuring the response force for a particular manipulation has allowed the free energies of various nucleic acid structures and configurations to be determined. Optical tweezers represent a class of single molecule experiments that allows the energies and structural dynamics of DNA to be probed up to and beyond the transition from the double helix to its melted single strands. These experiments are capable of high force resolution over a wide dynamic range. Additionally, these investigations may be compared with results obtained when the nucleic acids are in the presence of proteins or other binding ligands. These ligands may bind into the major or minor groove of the double helix, intercalate between bases or associate with an already melted single strand of DNA. By varying solution conditions and the pulling dynamics, energetic and dynamic information may be deduced about the mechanisms of binding to nucleic acids, providing insight into the function of proteins and the utility of drug treatments. © 2006 Wiley Periodicals, Inc. Biopolymers 85:154–168, 2007.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

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“…The gp32 protein coats the ssDNA produced by the primosome and is thought to be involved in the coordination of lagging strand synthesis (15). gp32 is made up of N-terminal, C-terminal, and core domains (16). The N-terminal domain (domain B for "basic") is involved in cooperative ssDNA binding.…”

mentioning

confidence: 99%

RNA Primer Handoff in Bacteriophage T4 DNA Replication

Nelson

Kumar

Benkovic

2008

Journal of Biological Chemistry

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In T4 phage, coordinated leading and lagging strand DNA synthesis is carried out by an eight-protein complex termed the replisome. The control of lagging strand DNA synthesis depends on a highly dynamic replisome with several proteins entering and leaving during DNA replication. Here we examine the role of single-stranded binding protein (gp32) in the repetitive cycles of lagging strand synthesis. Removal of the proteininteracting domain of gp32 results in a reduction in the number of primers synthesized and in the efficiency of primer transfer to the polymerase. We find that the primase protein is moderately processive, and this processivity depends on the presence of fulllength gp32 at the replication fork. Surprisingly, we find that an increase in the efficiency of primer transfer to the clamp protein correlates with a decrease in the dissociation rate of the primase from the replisome. These findings result in a revised model of lagging strand DNA synthesis where the primase remains as part of the replisome after each successful cycle of Okazaki fragment synthesis. A delay in primer transfer results in an increased probability of the primase dissociating from the replication fork. The interplay between gp32, primase, clamp, and clamp loader dictates the rate and efficiency of primer synthesis, polymerase recycling, and primer transfer to the polymerase. The T44 replisome has served as a highly useful model system for studying coupled DNA replication (1). The T4 replisome is made up of eight proteins, all of which have counterparts in more complex organisms such as Escherichia coli, Saccharomyces cerevisiae, and humans (2). DNA synthesis is carried out in a 5Ј to 3Ј direction by T4 DNA polymerase (gp43), which together with the clamp protein (gp45) makes up the holoenzyme complex (3). The holoenzyme can form through several different routes, all dependent on the activity of the clamp loader protein (gp44/62) (4, 5). The clamp loader is an AAAϩ protein that uses energy derived from ATP hydrolysis to chaperone the holoenzyme assembly process (6). The T4 primosome moves along the lagging strand DNA template in the 5Ј to 3Ј direction and is composed of a hexameric helicase (gp41) that unwinds the duplex DNA and an oligomeric primase (gp61) that synthesizes pentaribonucleotide primers at 5Ј-GTT and 5Ј-GCT sequences to initiate repetitive Okazaki fragment synthesis (7,8). The helicase is loaded onto the lagging strand template by the helicase loader protein (gp59) (9, 10). gp59 plays an additional role as a "gatekeeper" of the replisome by coordinating the assembly of the primosome with the initiation of leading strand DNA synthesis through a direct interaction with the leading strand polymerase (11-13). Finally, gp32 plays a central role in most aspects of DNA metabolism, including DNA replication (14). The gp32 protein coats the ssDNA produced by the primosome and is thought to be involved in the coordination of lagging strand synthesis (15). gp32 is made up of N-terminal, C-terminal, and core domains (16). The N...

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Domain Effects on the DNA-interactive Properties of Bacteriophage T4 Gene 32 Protein

Cited by 26 publications

References 25 publications

Modulation of T4 gene 32 protein DNA binding activity by the recombination mediator protein UvsY

Modulation of T4 gene 32 protein DNA binding activity by the recombination mediator protein UvsY

Mechanisms of DNA binding determined in optical tweezers experiments

RNA Primer Handoff in Bacteriophage T4 DNA Replication

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