Nucleic Acids Research, Vol 25, Issue 7 1450-1457, Copyright © 1997 by Oxford University Press
KA Eckert, SE Hile and PL Vargo
We have developed an in vitro DNA polymerase forward mutation assay using
damaged DNA templates that contain the herpes simplex virus type 1
thymidine kinase (HSV-tk) gene. The quantitative method uses complementary
strand hybridization to gapped duplex DNA molecules and chloramphenicol
selection. This design ensures exclusive analysis of mutations derived from
the DNA strand produced during in vitro synthesis. We have examined the
accuracy of DNA synthesis catalyzed by calf thymus polymerase
alpha-primase, polymerase beta and exonuclease- deficient Klenow
polymerase. Using unmodified DNA templates, polymerase beta displays a
unique specificity for the loss of two bases in a dinucleotide repeat
sequence within the HSV-tk locus. Treatment of the DNA template with
N-ethyl-N-nitrosourea resulted in a dose-dependent inhibition of DNA
synthesis concomitant with an increased mutation frequency. Similar
dose-response curves were measured for the three polymerases examined; thus
the identity of the DNA polymerase does not appear to affect the mutagenic
potency of ethyl lesions. The HSV-tk system is unique in that
damage-induced mutagenesis can be analyzed both quantitatively and
qualitatively in human cells, in bacterial cells and in in vitro DNA
synthesis reactions at a single target sequence.
ARTICLES
Development and use of an in vitro HSV-tk forward mutation assay to study eukaryotic DNA polymerase processing of DNA alkyl lesions
The Jake Gittlen Cancer Research Institute, The Pennsylvania State University College of Medicine, PO Box 850, Hershey, PA 17033, USA.
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