scholarly journals Development and use of an in vitro HSV-tk forward mutation assay to study eukaryotic DNA polymerase processing of DNA alkyl lesions

1997 ◽  
Vol 25 (7) ◽  
pp. 1450-1457 ◽  
Author(s):  
K. Eckert
Keyword(s):  
Dna Polymerase ◽  
Eukaryotic Dna ◽  
Mutation Assay ◽  
Forward Mutation

scholarly journals The TREX2 3′→ 5′ Exonuclease Physically Interacts with DNA Polymerase δ and Increases Its Accuracy

2002 ◽  
Vol 2 ◽  
pp. 275-281 ◽  
Author(s):  
Igor V. Shevelev ◽  
Kristijan Ramadan ◽  
Ulrich Hubscher
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Error Rates ◽  
High Fidelity ◽  
Replication Machinery ◽  
Dna Polymerase Δ ◽  
Pol I ◽  
Calf Thymus ◽  
Mutation Assay ◽  
Forward Mutation

Proofreading function by the 3′→ 5′ exonuclease of DNA polymerase δ (pol δ) is consistent with the observation that deficiency of the associated exonuclease can lead to a strong mutation phenotype, high error rates during DNA replication, and ultimately cancer. We have isolated pol δdfrom isotonic (pol δi) and detergent (pol δd) calf thymus extracts. Pol δdhad a 20-fold higher ratio of exonuclease to DNA polymerase than pol δi. This was due to the physical association of the TREX2 exonuclease to pol δd, which was missing from pol δi. Pol δdwas fivefold more accurate than pol δiunder error-prone conditions (1 μM dGTP and 20 dATP, dCTP, and dTTP) in a M13mp2 DNA forward mutation assay, and fourfold more accurate in an M13mp2T90 reversion assay. Under error-free conditions (20 μM each of the four dNTPs), however, both polymerases showed equal fidelity. Our data suggested that autonomous 3′→ 5′ exonucleases, such as TREX2, through its association with pol I can guarantee high fidelity under difficult conditions in the cell (e.g., imbalance of dNTPs) and can add to the accuracy of the DNA replication machinery, thus preventing mutagenesis.

Fatty acids selectively inhibit eukaryotic DNA polymerase activities in vitro

1996 ◽  
Vol 1308 (3) ◽  
pp. 256-262 ◽  
Author(s):  
Yoshiyuki Mizushina ◽  
Nobukazu Tanaka ◽  
Hisaaki Yagi ◽  
Takayoshi Kurosawa ◽  
Megumi Onoue ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Dna Polymerase ◽  
Eukaryotic Dna

scholarly journals Base Composition of Mononucleotide Runs Affects DNA Polymerase Slippage and Removal of Frameshift Intermediates by Mismatch Repair in Saccharomyces cerevisiae

2002 ◽  
Vol 22 (24) ◽  
pp. 8756-8762 ◽  
Author(s):  
Hana Gragg ◽  
Brian D. Harfe ◽  
Sue Jinks-Robertson
Keyword(s):  
Mismatch Repair ◽  
Dna Polymerase ◽  
Genome Stability ◽  
The Status ◽  
Nucleotide Insertion ◽  
Mismatch Recognition ◽  
Mutation Assay ◽  
Simple Repeats ◽  
Forward Mutation ◽  
Polymerase Slippage

ABSTRACT The postreplicative mismatch repair (MMR) system is important for removing mutational intermediates that are generated during DNA replication, especially those that arise as a result of DNA polymerase slippage in simple repeats. Here, we use a forward mutation assay to systematically examine the accumulation of frameshift mutations within mononucleotide runs of variable composition in wild-type and MMR-defective yeast strains. These studies demonstrate that (i) DNA polymerase slippage occurs more often in 10-cytosine/10-guanine (10C/10G) runs than in 10-adenine/10-thymine (10A/10T) runs, (ii) the MMR system removes frameshift intermediates in 10A/10T runs more efficiently than in 10C/10G runs, (iii) the MMR system removes −1 frameshift intermediates more efficiently than +1 intermediates in all 10-nucleotide runs, and (iv) the repair specificities of the Msh2p-Msh3p and Msh2p-Msh6p mismatch recognition complexes with respect to 1-nucleotide insertion/deletion loops vary dramatically as a function of run composition. These observations are relevant to issues of genome stability, with both the rates and types of mutations that accumulate in mononucleotide runs being influenced by the primary sequence of the run as well as by the status of the MMR system.

The In Vitro and In Vivo Genotoxicity of Benzocaine: A Brief Communication

2012 ◽  
Vol 31 (3) ◽  
pp. 222-227 ◽  
Author(s):  
William J. Brock ◽  
Thomas A. Bell
Keyword(s):  
Micronucleus Assay ◽  
The United States ◽  
Fda Approval ◽  
History Of ◽  
Good Laboratory ◽  
History Of Use ◽  
Mutation Assay ◽  
Forward Mutation

Benzocaine has a long history of use in human medicine. However, benzocaine also has been used in aquaculture with finfish for more than 40 years for sedating fish for marking, transport, surgery, and so on, although benzocaine does not have a current Food and Drug Administration (FDA) approval for this application in the United States. As part of a FDA approval for use as an animal drug, the genotoxicity of benzocaine was evaluated in the in vitro bacterial reverse mutation assay and the forward mutation assay and in vivo in the mouse micronucleus assay. These studies were conducted in compliance with Good Laboratory Practice regulations and according to Veterinary International Conference on Harmonisation guidelines. Based on the results of these studies, benzocaine was determined not to be genotoxic.

scholarly journals A sulphoquinovosyl diacylglycerol is a DNA polymerase epsilon inhibitor

2003 ◽  
Vol 370 (1) ◽  
pp. 299-305 ◽  
Author(s):  
Yoshiyuki MIZUSHINA ◽  
Xianai XU ◽  
Hitomi ASAHARA ◽  
Ryo TAKEUCHI ◽  
Masahiko OSHIGE ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerases ◽  
Specific Inhibitor ◽  
Immunosuppressive Agent ◽  
Biochemical Properties ◽  
Selective Inhibitor ◽  
Dna Polymerase Epsilon ◽  
Eukaryotic Dna ◽  
Polymerase Epsilon

Sulphoquinovosyl diacylglycerol (SQDG) was reported as a selective inhibitor of eukaryotic DNA polymerases α and β [Hanashima, Mizushina, Ohta, Yamazaki, Sugawara and Sakaguchi (2000) Jpn. J. Cancer Res. 91, 1073—1083] and an immunosuppressive agent [Matsumoto, Sahara, Fujita, Shimozawa, Takenouchi, Torigoe, Hanashima, Yamazaki, Takahashi, Sugawara et al. (2002) Transplantation 74, 261—267]. The purpose of this paper is to elucidate the biochemical properties of the inhibition more precisely. As expected, SQDG could inhibit the activities of mammalian DNA polymerases such as α, Δ, η and κ in vitro in the range of 2—5μM, and β and λ in vitro in the range of 20—45μM. However, SQDG could inhibit only mammalian DNA polymerases ∊ (pol ∊) activity at less than 0.04μM. SQDG bound more tightly to mammalian pol ∊ than the other mammalian polymerases tested. Moreover, SQDG could inhibit the activities of all the polymerases from animals such as fish and insect, but not of the polymerases from plant and prokaryotes. SQDG should, therefore, be called a mammalian pol ∊-specific inhibitor or animal polymerase-specific inhibitor. To our knowledge, this represents the first report about an inhibitor specific to mammalian pol ∊.

scholarly journals Purification of Drosophila DNA polymerase ζ by REV1 protein-affinity chromatography

2004 ◽  
Vol 382 (2) ◽  
pp. 535-543 ◽  
Author(s):  
Ryo TAKEUCHI ◽  
Masahiko OSHIGE ◽  
Makiyo UCHIDA ◽  
Gen ISHIKAWA ◽  
Kei-ichi TAKATA ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Biochemical Properties ◽  
Affinity Column ◽  
Protein Affinity ◽  
Pyrimidine Dimers ◽  
Multicellular Organisms ◽  
Mutation Assay

Studies on the biochemical properties of very-large-size eukaryotic DNA polymerases have been limited by the difficulty in obtaining sufficient purified forms of each enzyme. Our aim was to determine and elucidate the biochemical properties of one such polymerase, pol ζ (DNA polymerase ζ) from Drosophila melanogaster (Dmpol ζ). Using an REV1 (UV-revertible gene 1) protein-affinity column, we have isolated the enzyme directly from Drosophila embryos. Completely purified Dmpol ζ was found to have a molecular mass of approx. 240 kDa, and to be sensitive to aphidicolin and resistant to ddTTP (2′,3′-dideoxythymidine-5-triphosphate) and N-ethylmaleimide. The enzyme has a preference for poly(dA)/oligo(dT)10:1 as a template primer and has high processivity for DNA synthesis. Moreover, Dmpol ζ showed significantly higher fidelity compared with Rattus norvegicus DNA polymerase, an error-prone DNA polymerase, in an M13 forward mutation assay. The activities of bypassing pyrimidine dimers and (6-4) photoproducts and extending from mismatched primer-template termini in (6-4) photoproduct by Dmpol ζ were not detected. Drosophila REV7 interacted with Dmpol ζ in vitro, but did not influence the DNA synthesis activity of Dmpol ζ. The present study is the first report about characterization of purified pol ζ from multicellular organisms, and the second concerning the characterization of yeast pol ζ.

Sign in / Sign up

Export Citation Format

Share Document