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(Received for publication, January 24, 1997, and in revised form, March 19, 1997)
From the Department of Pathology and Laboratory Medicine,
Lineberger Comprehensive Cancer Center, University of North
Carolina, Chapel Hill, North Carolina 27599-7525
We constructed a double-stranded plasmid
containing a single cis,syn-cyclobutane thymine dimer
(T[c,s]T) 385 base pairs from the center of the SV40 origin of
replication. This circular DNA was replicated in vitro by
extracts from several types of human cells. The dimer was placed on the
leading strand template of the first replication fork to encounter the
lesion. Two-dimensional gel electrophoresis of replication
intermediates documented the transient arrest of the replication fork
by the dimer. Movement of the replication fork beyond the dimer was
recognized by the appearance of a single fork arc in DNA sequences
located between the T[c,s]T and the half-way point around the
circular template (180° from the origin). Upon completion of plasmid
replication, the T[c,s]T was detected by T4 endonuclease V in about
one-half (46 ± 9%) of the closed circular daughter molecules.
Our results demonstrate that extracts prepared from HeLa cells and
SV40-transformed human fibroblasts (SV80, IDH4), including a cell line
defective in nucleotide-excision repair (XPA), were competent for
leading strand DNA synthesis opposite the pyrimidine dimer and
replication fork bypass. In contrast, dimer bypass was severely
impaired in otherwise replication-competent extracts from two different
xeroderma pigmentosum variant cell lines.
Volume 272, Number 21,
Issue of May 23, 1997
pp. 13945-13954
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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