Eki T, Matsumoto T, Murakami Y, Hurwitz J
Graduate Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
J Biol Chem. 1992 Apr 15;267(11):7284-94.
The influence of DNA polymerase (pol) alpha and DNA primase on SV40 DNA replication was examined in both the monopolymerase and dipolymerase systems. The synthesis of oligoribonucleotides in the monopolymerase and dipolymerase systems, followed by pulse labeling with deoxynucleoside triphosphates, yielded short Okazaki fragments approximately 35 nucleotides in length that were chased into full-length Okazaki fragments with time. In the presence of activator 1 and proliferating cell nuclear antigen (PCNA), but no pol delta, these short fragments hardly increased in size with time. DNA fragments of similar size (approximately 35 nucleotides) were previously observed in SV40 replication reactions carried out with crude extracts of HeLa cells in the presence of antibodies directed against PCNA (Bullock, P. A., Seo, Y.S., and Hurwitz, J. (1991) Mol. Cell. Biol. 11, 2350-2361). Thus, the pol alpha-primase complex appears to act processively for only a short distance. At high levels of pol alpha and primase, both short and long DNA products were formed in both systems. In the presence of limiting amounts of pol alpha and excess primase, the monopolymerase system inefficiently yielded longer length Okazaki fragments than those formed with excess pol alpha and primase, whereas the dipolymerase system yielded both short and long DNA fragments. In the presence of limiting amounts of primase and excess pol alpha, long products were formed in both systems, and virtually no short products accumulated. Thus, the ratio between the polymerase and primer ends available controls the size of the nascent product DNA strands. We examined whether PCNA, the T4 phage-encoded gene product 45 (T4 gp45), and the Escherichia coli beta subunit of DNA polymerase III (dnaN gene product) supported SV40 DNA replication and the elongation of single-stranded DNA-binding protein-coated singly primed DNA in reactions catalyzed by pol delta, T4 DNA pol, and E. coli DNA pol III*, respectively. In the presence of T4 gp44/62 and T4 gp32 (but not human single-stranded DNA-binding protein isolated from HeLa cells), T4 DNA pol was weakly activated by PCNA and the beta subunit in lieu of T4 gp45 in the elongation of singly primed phi X174 DNA. However, the other systems were specific for their analogous auxiliary factors. This specificity indicates the importance of protein-protein interactions.
在单聚合酶和双聚合酶系统中研究了DNA聚合酶（pol）α和DNA引发酶对SV40 DNA复制的影响。在单聚合酶和双聚合酶系统中合成寡核糖核苷酸，随后用脱氧核苷三磷酸进行脉冲标记，产生了长度约为35个核苷酸的短冈崎片段，随着时间的推移这些片段会延伸为全长冈崎片段。在激活剂1和增殖细胞核抗原（PCNA）存在但没有pol δ的情况下，这些短片段的大小几乎不会随时间增加。先前在用针对PCNA的抗体处理的HeLa细胞粗提物进行的SV40复制反应中观察到了类似大小（约35个核苷酸）的DNA片段（布洛克，P.A.，徐，Y.S.，和赫维茨，J.（1991年）《分子细胞生物学》11，2350 - 2361）。因此，pol α - 引发酶复合物似乎仅在短距离内进行持续性作用。在高浓度的pol α和引发酶存在时，两个系统中都形成了短和长的DNA产物。在pol α含量有限而引发酶过量的情况下，单聚合酶系统产生的冈崎片段长度比pol α和引发酶过量时形成的片段更长，但效率较低，而双聚合酶系统产生了短和长的DNA片段。在引发酶含量有限而pol α过量的情况下，两个系统中都形成了长产物，几乎没有积累短产物。因此，可用的聚合酶与引物末端之间的比例控制着新生产物DNA链的大小。我们分别研究了PCNA、T4噬菌体编码的基因产物45（T4 gp45）以及DNA聚合酶III的大肠杆菌β亚基（dnaN基因产物）是否支持SV40 DNA复制以及在pol δ、T4 DNA聚合酶和大肠杆菌DNA聚合酶III*催化的反应中支持单链DNA结合蛋白包被的单引物DNA的延伸。在T4 gp44/62和T4 gp32存在（但不是从HeLa细胞中分离的人单链DNA结合蛋白）的情况下，在单引物φX174 DNA的延伸过程中，PCNA和β亚基代替T4 gp45对T4 DNA聚合酶有微弱的激活作用。然而，其他系统对其类似的辅助因子具有特异性。这种特异性表明了蛋白质 - 蛋白质相互作用的重要性。
