Membrane-associated DNase activity controlled by genes 46 and 47 of bacteriophage T4D and elevated DNase activity associated with the T4 DAS mutation
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/sup -/ and 47/sup -/ mutant-infected and 46/sup +/ 47/sup +/ 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/sup -/ 47/sup -/ mutant-infected cells digested native or heavily nicked Escherichia coli DNA to acid-soluble products three to four times slower than 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 47/sup -/ and 47/sup -/ mutants were present singly or together. NaClO/sub 4/, a chaotropic agent, released both gp46 and gp47 from 46/sup +/ 47/sup +/ membranes, as well as the DNase activity controlled by genes 46 and 47. DNA cellulose chromatography of proteins released from membranes by NaClO/sub 4/ 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 NaClO/sub 4/, and 30-fold more upon elution from DNA cellulose). T4 das mutations, which partially suppress the defective phenotype of 46/sup -/ and 47/sup -/ mutants, caused a considerable increase in in vitro DNase activity in both membrane and cytoplasmic fractions.
- Research Organization:
- Univ. of Rochester School of Medicine and Dentistry, NY
- OSTI ID:
- 5715123
- Journal Information:
- J. Virol.; (United States), Vol. 40:1
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
CELL MEMBRANES
ENZYME ACTIVITY
DNA
DECOMPOSITION
LETHAL MUTATIONS
GENETIC EFFECTS
BACTERIOPHAGES
CELL CULTURES
DNA REPLICATION
DNA-ASE
ESCHERICHIA COLI
EXPERIMENTAL DATA
GENE RECOMBINATION
GENES
LIQUID COLUMN CHROMATOGRAPHY
BACTERIA
BIOLOGICAL EFFECTS
CELL CONSTITUENTS
CHEMICAL REACTIONS
CHROMATOGRAPHY
DATA
ENZYMES
ESTERASES
HYDROLASES
INFORMATION
MEMBRANES
MICROORGANISMS
MUTATIONS
NUCLEIC ACID REPLICATION
NUCLEIC ACIDS
NUMERICAL DATA
ORGANIC COMPOUNDS
PARASITES
PHOSPHODIESTERASES
SEPARATION PROCESSES
VIRUSES
550300* - Cytology
550200 - Biochemistry