Georgaki A, Tuteja N, Sturzenegger B, Hübscher U
Department of Veterinary Biochemistry, University of Zürich-Irchel, Switzerland.
Nucleic Acids Res. 1994 Apr 11;22(7):1128-34. doi: 10.1093/nar/22.7.1128.
A DNA helicase from calf thymus, called DNA helicase F, copurified with replication protein A through several steps of purification including DEAE-Sephacel, hydroxyapatite and single stranded DNA cellulose. It is finally separated from replication protein A on FPLC Mono Q where the DNA helicase elutes after replication protein A. Characterization of the DNA helicase F by affinity labeling with [alpha 32P]ATP indicated that the enzyme has a catalytic subunit of 72 kDa. Gel filtration experiments suggested that DNA helicase F can exist both in a monomeric and an oligomeric form. The enzyme unwinds DNA in the 5'-->3' direction in relation to the strand it binds. All eight deoxyribonucleoside- and ribonucleosidetriphosphates could serve as an energy source. Testing a variety of DNA/DNA substrates demonstrated that the DNA helicase F preferentially unwinds very short substrates and is slightly stimulated by a single stranded 3'-tail. However, replication protein A allowed the DNA helicase to unwind much longer DNA substrates of up to 400 bases, indicating that the copurification of replication protein A with the DNA helicase F might be of functional relevance.
一种来自小牛胸腺的DNA解旋酶，称为DNA解旋酶F，它与复制蛋白A通过包括DEAE-琼脂糖凝胶、羟基磷灰石和单链DNA纤维素在内的几步纯化过程共同纯化。最终在FPLC Mono Q上从复制蛋白A中分离出来，在该柱上DNA解旋酶在复制蛋白A之后洗脱。用[α-32P]ATP进行亲和标记对DNA解旋酶F进行表征表明，该酶有一个72 kDa的催化亚基。凝胶过滤实验表明，DNA解旋酶F可以以单体和寡聚体形式存在。该酶相对于其结合的链以5'→3'方向解旋DNA。所有八种脱氧核糖核苷三磷酸和核糖核苷三磷酸都可以作为能量来源。对多种DNA/DNA底物进行测试表明，DNA解旋酶F优先解旋非常短的底物，并受到单链3'-尾的轻微刺激。然而，复制蛋白A使DNA解旋酶能够解旋长达400个碱基的长得多的DNA底物，这表明复制蛋白A与DNA解旋酶F的共同纯化可能具有功能相关性。
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