Nucleic Acids Research, Vol 24, Issue 19 3763-3770, Copyright © 1996 by Oxford University Press
K Nealon, ID Nicholl and MK Kenny
Base excision repair is one of the major mechanisms by which cells correct
damaged DNA. We have developed an in vitro assay for base excision repair
which is dependent on a uracil-containing DNA template. In this report, we
demonstrate the fractionation of a human cell extract into two required
components. One fraction was extensively purified and by several criteria
shown to be identical to DNA polymerase beta (Polbeta). Purified,
recombinant Polbeta efficiently substituted for this fraction. Escherichia
coli PolI, mammalian Poldelta and to a lesser extent Polalpha and epsilon
also functioned in this assay. We provide evidence that multiple
polymerases function in base excision repair in human cell extracts. A
neutralizing antibody to Polbeta, which inhibited repair synthesis
catalyzed by pure Polbeta by approximately 90%, only suppressed repair in
crude extracts by a maximum of approximately 70%. An inhibitor of Polbeta,
ddCTP, decreased base excision repair in crude extracts by approximately
50%, whereas the Polalpha/delta/epsilon inhibitor, aphidicolin, reduced the
reaction by approximately 20%. A combination of these chemical inhibitors
almost completely abolished repair synthesis. These data suggest that
Polbeta is the major base excision repair polymerase in human cells, but
that other polymerases also contribute to a significant extent.
ARTICLES
Characterization of the DNA polymerase requirement of human base excision repair
The Picower Institute for Medical Research, Manhasset, NY 11030, USA.
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