Kerr S M, Smith G L
Department of Pathology, University of Cambridge, UK.
Nucleic Acids Res. 1989 Nov 25;17(22):9039-50. doi: 10.1093/nar/17.22.9039.
Vaccinia virus gene SalF 15R potentially encodes a polypeptide of 63 kD which shares 30% amino acid identity with S. pombe and S. cerevisiae DNA ligases. DNA ligase proteins can be identified by incubation with alpha-(32P)ATP, resulting in the formation of a covalent DNA ligase-AMP adduct, an intermediate in the enzyme reaction. A novel radio-labelled polypeptide of approximately 61 kD appears in extracts from vaccinia virus infected cells after incubation with alpha-(32P)ATP. This protein is present throughout infection and is a DNA ligase as the radioactivity is discharged in the presence of either DNA substrate or pyrophosphate. DNA ligase assays show an increase in enzyme activity in cell extracts after vaccinia virus infection. A rabbit antiserum, raised against a bacterial fusion protein of beta-galactosidase and a portion of SalF 15R, immune-precipitates polypeptides of 61 and 54 kD from extracts of vaccinia virus-infected cells. This antiserum also immune-precipitates the novel DNA ligase-AMP adduct, thus proving that the observed DNA ligase is encoded by SalF 15R.
痘苗病毒基因SalF 15R可能编码一种63 kD的多肽，该多肽与粟酒裂殖酵母和酿酒酵母的DNA连接酶具有30%的氨基酸同一性。DNA连接酶蛋白可通过与α-(32P)ATP孵育来鉴定，这会导致形成共价DNA连接酶-AMP加合物，这是酶反应中的一个中间体。在用α-(32P)ATP孵育后，在痘苗病毒感染细胞的提取物中出现了一种约61 kD的新型放射性标记多肽。这种蛋白质在整个感染过程中都存在，并且是一种DNA连接酶，因为在存在DNA底物或焦磷酸的情况下放射性会释放。DNA连接酶分析表明，痘苗病毒感染后细胞提取物中的酶活性增加。一种针对β-半乳糖苷酶和SalF 15R一部分的细菌融合蛋白产生的兔抗血清，从痘苗病毒感染细胞的提取物中免疫沉淀出61 kD和54 kD的多肽。这种抗血清还免疫沉淀新型DNA连接酶-AMP加合物，从而证明观察到的DNA连接酶是由SalF 15R编码的。
