Membrane-associated DNase activity controlled by genes 46 and 47 of bacteriophage T4D and elevated DNase activity associated with the T4 das mutation
Membrane-associated DNase activity controlled by genes 46 and 47 of bacteriophage T4D and elevated DNase activity associated with the T4 das mutation
复制标题
膜相关 DNase 活性由噬菌体 T4D 的基因 46 和 47 控制，以及与 T4 das 突变相关的 DNase 活性升高
DOI：
10.1128/jvi.40.1.65-77.1981
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发表时间：
1981
影响因子：
5.4
通讯作者：
A. J. S. Wiberg
中科院分区：
文献类型：
--
作者：
Claudia;MICKELSON't;A. J. S. Wiberg
T4基因46和47中的致死性琥珀突变导致宿主DNA的不完全降解、噬菌体DNA合成的过早停滞、异常DNA复制中间体的积累和缺陷性重组。这些表型可以通过基因46和47控制DNA外切核酸酶的假设来解释，但是这种核酸酶的体外证明尚未报道。测定来自46和47 μ M感染的细胞和46+ 47+对照感染的细胞的膜和上清液级分中这些基因的蛋白产物的存在（即，gp 46和gp 47）和在体外将各种DNA底物降解为酸溶性产物的能力。这两种蛋白质仅在细胞膜上发现。从46- 47-mu感染细胞的膜部分消化天然或严重切口大肠杆菌DNA的酸溶性产品慢三至四倍，从控制感染细胞的膜部分。在细胞质组分中没有发现这种效应。无论46-和47-突变单独或一起存在，对膜中核酸酶活性的影响是相同的。离液剂NaClO 4从46+ 47+膜释放gp 46和gp 47，以及由基因46和47控制的DNA酶活性。用NaClO_4对膜蛋白进行DNA纤维素层析，结果表明，gp 46和gp 47与两种大肠杆菌的天然DNA结合。coli和T4.因此，gp 46和gp 47相对于总T4蛋白的总体富集为600倍（在膜中为10倍，在通过NaClO 4从膜释放时为2倍，在从DNA纤维素洗脱时为30倍）。T4 das突变部分抑制了46-和47-突变体的缺陷表型，导致细胞膜和细胞质组分中的体外DNA酶活性显著增加。大肠杆菌外切核酸酶I或V、内切核酸酶I或uvrA基因的UV内切核酸酶，或降低T4外切核酸酶A或T4基因43外切核酸酶的活性。
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.