Mickelson C, Wiberg J S
J Virol. 1981 Oct;40(1):65-77. doi: 10.1128/JVI.40.1.65-77.1981.
Lethal, amber mutations in T4 genes 46 and 47 cause incomplete degradation of host DNA, premature arrest of phage DNA synthesis, accumulation of abnormal DNA replication intermediates, and defective recombination. These phenotypes can be explained by the hypothesis that genes 46 and 47 control a DNA exonuclease, but in vitro demonstration of such a nuclease has not yet been reported. Membrane and supernatant fractions from 46- and 47- mutant-infected and 46+ 47+ control-infected cells were assayed for the presence of the protein products of these genes (i.e., gp46 and gp47) and for the ability to degrade various DNA substrates to acid-soluble products in vitro. The two proteins were found only on membranes. The membrane fraction from 46- 47- mutant-infected cells digested native or heavily nicked Escherichia coli DNA to acid-soluble products three to four times slower that the membrane fraction from control-infected cells. No such effect was found in the cytoplasmic fractions. The effect on nuclease activity in membranes was the same whether 46- and 47- mutations were present singly or together. NaClO4, a chaotropic agent, released both gp46 and gp47 from 46+ 47+ membranes, as well as the DNase activity controlled by genes 46 and 47. DNA cellulose chromatography of proteins released from membranes by NaClO4 showed that gp46 and gp47 bound to the native DNAs of both E. coli and T4. Thus, the overall enrichment of gp46 and gp47 relative to total T4 protein was 600-fold (10-fold in membranes, 2-fold more upon release from membranes by NaClO4, and 30-fold more upon elution from DNA cellulose). T4 das mutations, which partially suppress the defective phenotype of 46- and 47- mutants, caused a considerable increase in vitro DNase activity in both membrane and cytoplasmic fractions, We obtained evidence that the das+ gene does not function to inhibit E. coli exonuclease I or V, endonuclease I, or the UV endonuclease of gene uvrA or to decrease the activity of T4 exonuclease A or the T4 gene 43 exonuclease.
T4基因46和47中的致死性琥珀突变会导致宿主DNA降解不完全、噬菌体DNA合成过早停滞、异常DNA复制中间体积累以及重组缺陷。这些表型可以用基因46和47控制一种DNA外切核酸酶的假说来解释，但尚未有该核酸酶的体外证明报道。对感染46和47突变体以及感染46 + 47 +对照的细胞的膜和上清液部分进行检测，以确定这些基因的蛋白质产物（即gp46和gp47）是否存在，以及体外将各种DNA底物降解为酸溶性产物的能力。仅在膜上发现了这两种蛋白质。感染46 - 47 -突变体的细胞的膜部分将天然或严重切口的大肠杆菌DNA消化为酸溶性产物的速度比感染对照的细胞的膜部分慢三到四倍。在细胞质部分未发现这种效应。无论46和47突变是单独存在还是同时存在，对膜中核酸酶活性的影响都是相同的。离液剂高氯酸钠从46 + 47 +膜中释放出gp46和gp47，以及由基因46和47控制的DNase活性。用高氯酸钠从膜中释放的蛋白质进行DNA纤维素层析表明，gp46和gp47与大肠杆菌和T4的天然DNA结合。因此，相对于总T4蛋白，gp46和gp47的总体富集倍数为600倍（在膜中为10倍，从膜中用高氯酸钠释放后增加2倍，从DNA纤维素上洗脱后增加30倍）。部分抑制46和47突变体缺陷表型的T4 das突变导致膜和细胞质部分的体外DNase活性显著增加。我们获得的证据表明，das +基因的功能不是抑制大肠杆菌外切核酸酶I或V、核酸内切酶I或uvrA基因的UV核酸内切酶，也不是降低T4外切核酸酶A或T4基因43外切核酸酶的活性。
