Ruyechan W T
Department of Biochemistry, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
J Virol. 1988 Mar;62(3):810-7. doi: 10.1128/JVI.62.3.810-817.1988.
The major herpes simplex virus DNA-binding protein, designated ICP8, binds tightly to single-stranded DNA and is required for replication of viral DNA. The sensitivity of the DNA-binding activity of ICP8 to the action of the sulfhydryl reagent N-ethylmaleimide has been examined by using nitrocellulose filter-binding and agarose gel electrophoresis assays. Incubation of ICP8 with N-ethylmaleimide results in a rapid loss of DNA-binding activity. Preincubation of ICP8 with single-stranded DNA markedly inhibits this loss of binding activity. These results imply that a free sulfhydryl group is involved in the interaction of ICP8 with single-stranded DNA and that this sulfhydryl group becomes less accessible to the environment upon binding. Agarose gel electrophoretic analysis of the binding interaction in the presence and absence of N-ethylmaleimide indicates that the cooperative binding exhibited by ICP8 is lost upon treatment with this reagent but that some residual noncooperative binding may remain. This last result was confirmed by equilibrium dialysis experiments with the 32P-labeled oligonucleotide dT10 and native and N-ethylmaleimide-treated ICP8.
主要的单纯疱疹病毒DNA结合蛋白，命名为ICP8，能紧密结合单链DNA，是病毒DNA复制所必需的。通过使用硝酸纤维素滤膜结合和琼脂糖凝胶电泳分析，研究了ICP8的DNA结合活性对巯基试剂N - 乙基马来酰亚胺作用的敏感性。将ICP8与N - 乙基马来酰亚胺一起温育会导致DNA结合活性迅速丧失。将ICP8与单链DNA预温育可显著抑制这种结合活性的丧失。这些结果表明，一个游离的巯基参与了ICP8与单链DNA的相互作用，并且该巯基在结合后变得较难与周围环境接触。在有和没有N - 乙基马来酰亚胺存在的情况下对结合相互作用进行琼脂糖凝胶电泳分析表明，ICP8所表现出的协同结合在用该试剂处理后丧失，但可能仍存在一些残余的非协同结合。用32P标记的寡核苷酸dT10以及天然的和经N - 乙基马来酰亚胺处理的ICP8进行平衡透析实验证实了这一最终结果。
