T4 replication: What does “processivity” really mean?

Joyce, Catherine M.

doi:10.1073/pnas.0402850101

Cited by 7 publications

(5 citation statements)

References 13 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…There is now mounting experimental evidence for such dissociationrebinding events as proposed in the nonspecific-bindingfacilitated diffusion model (Halford and Marko, 2004;Hannon et al, 1986;Houtsmuller et al, 1999;Phair and Misteli, 2000;Lever et al, 2000;Misteli et al, 2000;Gowers and Halford, 2003). Even the archetypical processive proteins, replicative DNA polymerases, have now been shown to undergo rapid exchange during replisome-mediated DNA replication (Yang et al, 2004;Joyce, 2004). In addition, Jeltsch and Pingoud (1998) presented experimental evidence that DNA ends are reflecting to EcoRV, supporting the assumption (Eq.…”

Section: Comparison With Experimental Datamentioning

confidence: 69%

A Model for the Mediation of Processivity of DNA-Targeting Proteins by Nonspecific Binding: Dependence on DNA Length and Presence of Obstacles

Zhou

2005

Biophysical Journal

View full text Add to dashboard Cite

A physical and mathematical model is presented to explain processivity of proteins on DNA. In this model, a DNA-targeting protein such as a restriction enzyme can diffuse to the DNA surface and nonspecifically bind to it. Once on the DNA surface it will either move along the DNA or equilibrate with the surrounding region. Owing to the nonspecific binding, the search for a specific site on the DNA occurs in a reduced dimensionality, and the protein appears processive when moving from one specific site to another. The simplest version of this nonspecific-binding-facilitated diffusion model is solved and the results quantitatively explain experimentally observed dependence of the processivity ratio on the intervening DNA length between two specific sites.

show abstract

Section: Comparison With Experimental Datamentioning

confidence: 69%

A Model for the Mediation of Processivity of DNA-Targeting Proteins by Nonspecific Binding: Dependence on DNA Length and Presence of Obstacles

Zhou

2005

Biophysical Journal

View full text Add to dashboard Cite

show abstract

“…Although researchers have known for years that tethering of the T4 DNA polymerase in holoenzyme complexes increases both polymerase and exonuclease activities, Yang et al (2004) observed that the primer-template appears to transfer between two tethered polymerases during proofreading. Thus, apparent continuous replication by the tethered T4 DNA polymerase may involve coordinated replication by two DNA polymerases tethered to the same clamp ( Joyce, 2004 ). Tethering, however, does not fully compensate for differences in intrinsic processivity as explained below.…”

Section: Resultsmentioning

confidence: 99%

Engineering processive DNA polymerases with maximum benefit at minimum cost

2014

View full text Add to dashboard Cite

DNA polymerases need to be engineered to achieve optimal performance for biotechnological applications, which often require high fidelity replication when using modified nucleotides and when replicating difficult DNA sequences. These tasks are achieved for the bacteriophage T4 DNA polymerase by replacing leucine with methionine in the highly conserved Motif A sequence (L412M). The costs are minimal. Although base substitution errors increase moderately, accuracy is maintained for templates with mono- and dinucleotide repeats while replication efficiency is enhanced. The L412M substitution increases intrinsic processivity and addition of phage T4 clamp and single-stranded DNA binding proteins further enhance the ability of the phage T4 L412M-DNA polymerase to replicate all types of difficult DNA sequences. Increased pyrophosphorolysis is a drawback of increased processivity, but pyrophosphorolysis is curbed by adding an inorganic pyrophosphatase or divalent metal cations, Mn2+ or Ca2+. In the absence of pyrophosphorolysis inhibitors, the T4 L412M-DNA polymerase catalyzed sequence-dependent pyrophosphorolysis under DNA sequencing conditions. The sequence specificity of the pyrophosphorolysis reaction provides insights into how the T4 DNA polymerase switches between nucleotide incorporation, pyrophosphorolysis and proofreading pathways. The L-to-M substitution was also tested in the yeast DNA polymerases delta and alpha. Because the mutant DNA polymerases displayed similar characteristics, we propose that amino acid substitutions in Motif A have the potential to increase processivity and to enhance performance in biotechnological applications. An underlying theme in this chapter is the use of genetic methods to identify mutant DNA polymerases with potential for use in current and future biotechnological applications.

show abstract

“…Yang et al ( 36 ) demonstrated that T4 DNA polymerases exchange during DNA replication and that this exchange requires the clamp. Since the clamp can potentially bind the replicating DNA polymerase and a ‘spare’ DNA polymerase, incorporation of a wrong nucleotide may lead to dissociation of the replicating DNA polymerase and then the spare DNA polymerase forms an exonuclease complex with the mismatched DNA ( 20 , 37 ). The role of the clamp then is to provide a locally high concentration of spare DNA polymerases at replication forks for exonucleolytic proofreading.…”

Section: Discussionmentioning

confidence: 99%

DNA polymerase proofreading: active site switching catalyzed by the bacteriophage T4 DNA polymerase

Silva

Reha-Krantz

2007

Nucleic Acids Research

View full text Add to dashboard Cite

DNA polymerases achieve high-fidelity DNA replication in part by checking the accuracy of each nucleotide that is incorporated and, if a mistake is made, the incorrect nucleotide is removed before further primer extension takes place. In order to proofread, the primer-end must be separated from the template strand and transferred from the polymerase to the exonuclease active center where the excision reaction takes place; then the trimmed primer-end is returned to the polymerase active center. Thus, proofreading requires polymerase-to-exonuclease and exonuclease-to-polymerase active site switching. We have used a fluorescence assay that uses differences in the fluorescence intensity of 2-aminopurine (2AP) to measure the rates of active site switching for the bacteriophage T4 DNA polymerase. There are three findings: (i) the rate of return of the trimmed primer-end from the exonuclease to the polymerase active center is rapid, >500 s−1; (ii) T4 DNA polymerase can remove two incorrect nucleotides under single turnover conditions, which includes presumed exonuclease-to-polymerase and polymerase-to-exonuclease active site switching steps and (iii) proofreading reactions that initiate in the polymerase active center are not intrinsically processive.

show abstract

T4 replication: What does “processivity” really mean?

Cited by 7 publications

References 13 publications

A Model for the Mediation of Processivity of DNA-Targeting Proteins by Nonspecific Binding: Dependence on DNA Length and Presence of Obstacles

A Model for the Mediation of Processivity of DNA-Targeting Proteins by Nonspecific Binding: Dependence on DNA Length and Presence of Obstacles

Engineering processive DNA polymerases with maximum benefit at minimum cost

DNA polymerase proofreading: active site switching catalyzed by the bacteriophage T4 DNA polymerase

Contact Info

Product

Resources

About