Nucleic Acids Research, Vol 26, Issue 16 3729-3738, Copyright © 1998 by Oxford University Press
L Islas, CF Fairley and WF Morgan
DNA polymerases catalyze the synthesis of DNA using a continuous
uninterrupted template strand. However, it has been shown that a 3'--
>5' exonuclease-deficient form of the Klenow fragment of Escherichia
coli DNA polymerase I as well as DNA polymerase of Thermus aquaticus can
synthesize DNA across two unlinked DNA templates. In this study, we used an
oligonucleotide-based assay to show that discontinuous DNA synthesis was
present in HeLa cell extracts. DNA synthesis inhibitor studies as well as
fractionation of the extracts revealed that most of the discontinuous DNA
synthesis was attributable to DNA polymerase alpha. Additionally,
discontinuous DNA synthesis could be eliminated by incubation with an
antibody that specifically neutralized DNA polymerase alpha activity. To
test the relative efficiency of each nuclear DNA polymerase for
discontinuous synthesis, equal amounts (as measured by DNA polymerase
activity) of DNA polymerases alpha, beta, delta (+/- PCNA) and
straightepsilon (+/- PCNA) were used in the discontinuous DNA synthesis
assay. DNA polymerase alpha showed the most discontinuous DNA synthesis
activity, although small but detectable levels were seen for DNA
polymerases delta (+PCNA) and straightepsilon (- PCNA). Klenow fragment and
DNA polymerase beta showed no discontinuous DNA synthesis, although at much
higher amounts of each enzyme, discontinuous synthesis was seen for both.
Discontinuous DNA synthesis by DNA polymerase alpha was seen with
substrates containing 3 and 4 bp single-strand stretches of
complementarity; however, little synthesis was seen with blunt substrates
or with 1 bp stretches. The products formed from these experiments are
structurally similar to that seen in vivo for non-homologous end joining in
eukaryotic cells. These data suggest that DNA polymerase alpha may be able
to rejoin double- strand breaks in vivo during replication.
ARTICLES
DNA synthesis on discontinuous templates by human DNA polymerases: implications for non-homologous DNA recombination
Department of Radiation Oncology, University of California, San Francisco, CA 94143-0750, USA. aislas@lbl.gov
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