IDENTIFICATION OF A PROTEIN LINKED TO ENDS OF ADENOVIRUS DNA
IDENTIFICATION OF A PROTEIN LINKED TO ENDS OF ADENOVIRUS DNA
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DOI：
10.1016/0092-8674(77)90045-9
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发表时间：
1977-01-01
期刊：
影响因子：
64.5
通讯作者：
ROBINSON, AJ
中科院分区：
文献类型：
--
作者：
REKOSH, DMK;RUSSELL, WC;ROBINSON, AJ
用电镜、放射化学标记和分析超电泳对人腺病毒的DNA-蛋白复合物进行了进一步的表征。复合物的制备物含有大百分比的DNA形式，其为环状或寡聚体，并且通过在含有4 M氯化胍的CsCl梯度中进行分析性超离心，容易与链霉蛋白酶处理的腺病毒DNA的制备物区分开。使用博尔顿和亨特试剂在体外用125 I碘化蛋白质组分，标记蛋白质的SDS[十二烷基硫酸钠]-聚丙烯酰胺凝胶分析表明其具有55，000道尔顿的表观MW。用32 PO 4标记的DNA-蛋白质复合物的DNA酶I消化导致释放32 PO 4标记的蛋白质，该蛋白质即使在1%SDS和1%巯基乙醇中煮沸后仍保持标记。随后用蛇毒磷酸二酯酶消化该实体导致释放32 P4标记的5“-磷酸脱氧核苷酸。用Eco R1消化DNA-蛋白质复合物并分析分离的限制性片段，表明蛋白质存在于每个末端片段上。显然，分子的每个5“端可能通过共价键直接连接有55，000道尔顿的蛋白质。该蛋白在DNA复制过程中通过促进子代链的引发，允许DNA的5“末端被复制而明显起作用。
A DNA-protein complex from human adenoviruses were further characterized by EM, radiochemical labeling and analytical ultracentrifugation. Preparations of the complex contain a large percentage of forms of DNA which are circular or oligomeric and are readily distinguishable from preparations of Pronase-treated adenovirus DNA by analytical ultracentrifugation in CsCl gradients containing 4 M guanidinium chloride. The protein component was iodinated in vitro with 125I using Bolton and Hunter reagent, and SDS[sodium dodecyl sulfate]-polyacrylamide gel analysis of the labeled protein indicates that it has an apparent MW of 55,000 daltons. DNase I digestion of the DNA-protein complex labeled with 32PO4 results in release of a 32PO4-labeled protein which remains labeled even after boiling in 1% SDS and 1% mercaptoethanol. Subsequent digestion of this entity with snake venom phosphodiesterase leads to release of 32P4-labeled 5''-phosphate deoxynucleotides. Digestion of the DNA-protein complex with Eco R1 and analysis of the isolated restriction fragments indicates that the protein is present on each terminal fragment. There apparently is a protein of 55,000 daltons directly attached to each 5'' end of molecule probably via a covalent linkage. The protein apparently functions during DNA replication by facilitating priming of the progeny strands, allowing the 5'' ends of the DNA to be replicated.