Replication fork bypass of a pyrimidine dimer blocking leading strand DNA synthesis.
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 degrees 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.
Cordeiro-Stone
M
M
Department of Pathology and Laboratory Medicine, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-7525, USA. uncmcs@med.unc.edu
Zaritskaya
L S
LS
Price
L K
LK
Kaufmann
W K
WK
D002384:Catalysis; D004249:DNA Damage; D004261:DNA Replication; D004262:DNA Restriction Enzymes / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D008957:Models, Genetic; D010957:Plasmids / Q000378:metabolism; D011740:Pyrimidine Dimers / Q000378:metabolism*; D013539:Simian virus 40; D014983:Xeroderma Pigmentosum / Q000235:genetics
D011740:Pyrimidine Dimers; D004262:DNA Restriction Enzymes