TY - JOUR
T1 - Uncoupling of SV40 tsA replicon activation from DNA chain elongation by temperature shifts and aphidicolin arrest
AU - Dinter-Gottlieb, Gail
AU - Kaufmann, Gabriel
N1 - Funding Information:
The gift of aphidicolin from S. Ikegami and from A.H. Todd is gratefully acknowledged. We thank D. Billen, S. Eisenberg, S. Lavi and E. Winocour for discussions. This study was supported by grants 1684 and 2432 from the United States-Israel Binational Science Foundation, Jerusalem. G.K. was an incumbent of the Alan Dixon Career Development Chair.
PY - 1982/1/22
Y1 - 1982/1/22
N2 - To synchronize SV40 replicons, simian cells infected with a tsA mutant were restricted at 40°, to complete ongoing replication and returned to 32°, to activate new replicons in the presence of the DNA chain elongation inhibitor aphidicolin. Upon further incubation at 40° without the drug, 3H-dT was incorporated into SV40 FI DNA, almost to the extent seen with cells recovered in the absence of the drug. To determine whether DNA synthesis would begin from the origin, following the temperature-shifts-aphidicolin regimen, chains subsequently pulse-labeled with (α-32P in dGTP isolated nuclei were analyzed for size distribution and genomic location. These chains reached up to 300-400 nucleotides in size, unlike the control which featured comparable amounts of label in long chains and Okazaki pieces. The nascent DNA of the drug-treated system could be chased into longer chains, indicating that it was a replicative intermediate; and it hybridized preferentially to an origin proximal fragment of AtuI restricted SV4O DNA, demonstrating partial replicon synchronization.The data prove that T-antigen activates the SV40 replicon independent of DNA chain elongation and suggest means to study the mechanism of DNA chain priming at the origin.
AB - To synchronize SV40 replicons, simian cells infected with a tsA mutant were restricted at 40°, to complete ongoing replication and returned to 32°, to activate new replicons in the presence of the DNA chain elongation inhibitor aphidicolin. Upon further incubation at 40° without the drug, 3H-dT was incorporated into SV40 FI DNA, almost to the extent seen with cells recovered in the absence of the drug. To determine whether DNA synthesis would begin from the origin, following the temperature-shifts-aphidicolin regimen, chains subsequently pulse-labeled with (α-32P in dGTP isolated nuclei were analyzed for size distribution and genomic location. These chains reached up to 300-400 nucleotides in size, unlike the control which featured comparable amounts of label in long chains and Okazaki pieces. The nascent DNA of the drug-treated system could be chased into longer chains, indicating that it was a replicative intermediate; and it hybridized preferentially to an origin proximal fragment of AtuI restricted SV4O DNA, demonstrating partial replicon synchronization.The data prove that T-antigen activates the SV40 replicon independent of DNA chain elongation and suggest means to study the mechanism of DNA chain priming at the origin.
UR - http://www.scopus.com/inward/record.url?scp=0020073341&partnerID=8YFLogxK
U2 - 10.1093/nar/10.2.763
DO - 10.1093/nar/10.2.763
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AN - SCOPUS:0020073341
SN - 0305-1048
VL - 10
SP - 763
EP - 773
JO - Nucleic Acids Research
JF - Nucleic Acids Research
IS - 2
ER -