Coffman F D, Fresa K L, Hameed M, Cohen S
Department of Pathology, Hahnemann University, Philadelphia, Pennsylvania 19102.
J Cell Biochem. 1993 Feb;51(2):157-64. doi: 10.1002/jcb.240510207.
Isolated cell nuclei were used as the source of template DNA to investigate the role of a cytosolic aprotinin-binding protein (ADR) in the initiation of eukaryotic DNA replication. Computerized image cytometry demonstrated that the DNA content of individual nuclei increased significantly following incubation with ADR-containing preparations, and the extent of DNA synthesis is consistent with that allowed by the limiting concentration of dTTP. Thus, dTTP incorporation into isolated nuclei represents DNA synthesis and not parent strand repair. We found that dTTP incorporation into the isolated nuclei is dependent on DNA polymerase alpha (a principal polymerase in DNA replication) but that DNA polymerase beta (a principal polymerase in DNA repair processes) does not play a significant role in this system. Finally, neither aprotinin nor a previously described cytosolic ADR inhibitor can block the replication of nuclease-treated calf thymus DNA, while both strongly inhibit replication of DNA in isolated nuclei. This result, coupled with the relative ineffectiveness of nuclease-treated DNA compared with nuclear DNA to serve as a replicative template in this assay, argues against a significant contribution from repair or synthesis which initiates at a site of DNA damage. These data indicate that ADR-mediated incorporation of 3H-dTTP into isolated nuclei results from DNA replicative processes that are directly relevant to in vivo S phase events.
分离的细胞核被用作模板DNA的来源，以研究一种胞质抑肽酶结合蛋白（ADR）在真核生物DNA复制起始中的作用。计算机化图像细胞术表明，与含ADR的制剂孵育后，单个细胞核的DNA含量显著增加，且DNA合成的程度与dTTP的极限浓度所允许的程度一致。因此，dTTP掺入分离的细胞核代表DNA合成而非亲本链修复。我们发现，dTTP掺入分离的细胞核依赖于DNA聚合酶α（DNA复制中的主要聚合酶），而DNA聚合酶β（DNA修复过程中的主要聚合酶）在该系统中不起重要作用。最后，抑肽酶和先前描述的胞质ADR抑制剂均不能阻断核酸酶处理的小牛胸腺DNA的复制，而二者均强烈抑制分离细胞核中DNA的复制。这一结果，再加上与核DNA相比，核酸酶处理的DNA在该测定中作为复制模板的相对无效性，反对了在DNA损伤位点起始的修复或合成的显著贡献。这些数据表明，ADR介导的3H-dTTP掺入分离的细胞核是由与体内S期事件直接相关的DNA复制过程导致的。
