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(Received for publication, January 8, 1997, and in revised form, February 18, 1997)
From the Department of Biochemistry and Molecular Biology, Wright
State University School of Medicine, Dayton, Ohio 45435
Using a synthetic telomere DNA template and whole
cell extracts, we have identified proteins capable of synthesizing the
telomere complementary strand. Synthesis of the complementary strand
required a DNA template consisting of 10 repeats of the human telomeric sequence d(TTAGGG) and deoxy- and ribonucleosidetriphosphates and was
inhibited by neutralizing antibodies to DNA polymerase
Volume 272, Number 18,
Issue of May 2, 1997
pp. 11678-11681
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
. No evidence
for RNA-independent synthesis of the lagging strand was observed,
suggesting that a stable DNA secondary structure capable of priming the
lagging strand is unlikely. Purified DNA polymerase /primase was
capable of catalyzing synthesis of the lagging strand with the same
requirements as those observed in crude cell extracts. A ladder of
products was observed with an interval of six bases, suggesting a
unique RNA priming site and site-specific pausing or dissociation of
polymerase on the d(TTAGGG)10 template. Removal
of the RNA primers was observed upon the addition of purified RNase HI.
By varying the input rNTP, the RNA priming site was determined to be
opposite the 3
thymidine nucleotide generating a five-base RNA primer
with the sequence 5-AACCC. The addition of UTP did not increase the
efficiency of priming and extension, suggesting that the five-base RNA
primer is sufficient for extension with dNTPs by DNA polymerase .
This represents the first experimental evidence for RNA priming and DNA
extension as the mechanism of mammalian telomeric lagging strand
replication.
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