Salt Dependent Binding of T4 Gene 32 Protein to Single and Double-stranded DNA: Single Molecule Force Spectroscopy Measurements

Pant, Kiran; Karpel, Richard L.; Rouzina, Ioulia; Williams, Mark C.

doi:10.1016/j.jmb.2005.03.065

Cited by 79 publications

(127 citation statements)

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“…Similar to other ssDNA-binding proteins, it is thought that ssDNA binds within the cleft of the oligonucleotide/oligosaccharide binding fold (OB-fold) via contacts with positively charged and aromatic residues (10). Proteolytic fragments of gp32 of phage T4 and E. coli SSB protein, lacking the C-terminal portions of these proteins were reported to bind ssDNA with higher affinity than the full-length protein (12)(13)(14)25). A similar phenomenon has been described for HMG (High Mobility Group) proteins found in chromatin (26).…”

Section: Discussionmentioning

confidence: 75%

Essential Residues in the C Terminus of the Bacteriophage T7 Gene 2.5 Single-stranded DNA-binding Protein

Marintcheva¹,

Hamdan²,

Lee³

et al. 2006

Journal of Biological Chemistry

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Gene 2.5 of bacteriophage T7 encodes a single-stranded DNA (ssDNA)-binding protein (gp2.5) that is an essential component of the phage replisome. Similar to other prokaryotic ssDNAbinding proteins, gp2.5 has an acidic C terminus that is involved in protein-protein interactions at the replication fork and in modulation of the ssDNA binding properties of the molecule. We have used genetic and biochemical approaches to identify residues critical for the function of the C terminus of gp2.5. The presence of an aromatic residue in the C-terminal position is essential for gp2.5 function. Deletion of the C-terminal residue, phenylalanine, is detrimental to its function, as is the substitution of this residue with non-aromatic amino acids. Placing the C-terminal phenylalanine in the penultimate position also results in loss of function. Moderate shortening of the length of the acidic portion of the C terminus is tolerated when the aromatic nature of the C-terminal residue is preserved. Gradual removal of the acidic C terminus of gp2.5 results in a higher affinity for ssDNA and a decreased ability to interact with T7 DNA polymerase/thioredoxin. The replacement of the charged residues in the C terminus with neutral amino acids abolishes gp2.5 function. Our data show that both the C-terminal aromatic residue and the overall acidic charge of the C terminus of gp2.5 are critical for its function.

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Section: Discussionmentioning

confidence: 75%

Essential Residues in the C Terminus of the Bacteriophage T7 Gene 2.5 Single-stranded DNA-binding Protein

Marintcheva¹,

Hamdan²,

Lee³

et al. 2006

Journal of Biological Chemistry

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“…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. A typical stretching/ relaxation cycle requires about 2 minutes.…”

Section: Figurementioning

confidence: 99%

“…To determine the equilibrium melting force, F eq , a series of time-dependent experiments have been performed. [100][101][102][103] The DNA-protein complex may be stretched into the overstretching plateau, specifically to a length of 0.42 nm per base pair. Alternatively, the DNA may be stretched, and then relaxed back to a length of 0.42 nm per base pair.…”

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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“…Using optical tweezers, the Williams group studied the effects of the T4 gp32 and the T7 gp2.5 ssDNA binding proteins on the melting behavior of duplex DNA (83)(84)(85)97). They used these data to understand the mechanisms of interaction between SSBs and DNA.…”

Section: Wwwannualreviewsorg • Single-molecule Studies Of Replicatimentioning

confidence: 99%

Single-Molecule Studies of the Replisome

Oijen

Loparo

2010

Annu. Rev. Biophys.

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Replication of DNA is carried out by the replisome, a multiprotein complex responsible for the unwinding of parental DNA and the synthesis of DNA on each of the two DNA strands. The impressive speed and processivity with which the replisome duplicates DNA are a result of a set of tightly regulated interactions between the replication proteins. The transient nature of these protein interactions makes it challenging to study the dynamics of the replisome by ensemble-averaging techniques. This review describes single-molecule methods that allow the study of individual replication proteins and their functioning within the replisome. The ability to mechanically manipulate individual DNA molecules and record the dynamic behavior of the replisome while it duplicates DNA has led to an improved understanding of the molecular mechanisms underlying DNA replication.

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Salt Dependent Binding of T4 Gene 32 Protein to Single and Double-stranded DNA: Single Molecule Force Spectroscopy Measurements

Cited by 79 publications

References 50 publications

Essential Residues in the C Terminus of the Bacteriophage T7 Gene 2.5 Single-stranded DNA-binding Protein

Essential Residues in the C Terminus of the Bacteriophage T7 Gene 2.5 Single-stranded DNA-binding Protein

Mechanisms of DNA binding determined in optical tweezers experiments

Single-Molecule Studies of the Replisome

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