Dinter-Gottlieb G, Kaufmann G
Nucleic Acids Res. 1982 Jan 22;10(2):763-73. doi: 10.1093/nar/10.2.763.
To synchronize SV40 replicons, simian cells infected with a tsA mutant were restricted at 40 degrees, to complete ongoing replication and returned to 32 degrees, to activate new replicons in the presence of the DNA chain elongation inhibitor aphidicolin. Upon further incubation at 40 degrees 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 (alpha-32p)dGTP in 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 SV40 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.
为了同步SV40复制子，将感染tsA突变体的猴细胞在40℃下培养，以完成正在进行的复制，然后回到32℃，在DNA链延伸抑制剂阿非迪霉素存在的情况下激活新的复制子。在无药物的情况下于40℃进一步孵育后，3H-dT掺入SV40 FI DNA中，掺入程度几乎与在无药物情况下回收的细胞中所见程度相同。为了确定在温度变化-阿非迪霉素处理方案之后DNA合成是否会从起始点开始，对随后在分离细胞核中用（α-32p）dGTP进行脉冲标记的链进行大小分布和基因组定位分析。这些链的大小可达300 - 400个核苷酸，这与对照不同，对照中长链和冈崎片段中的标记量相当。药物处理系统的新生DNA可以追踪到更长的链，表明它是复制中间体；并且它优先与AtuI限制性SV40 DNA的起始点近端片段杂交，证明了部分复制子同步。数据证明T抗原独立于DNA链延伸激活SV40复制子，并提出了研究起始点处DNA链引发机制的方法。
