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Membrane-associated DNase activity controlled by genes 46 and 47 of bacteriophage T4D and elevated DNase activity
Abstract:
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.
Insights
Mutations in T4 genes 46 and 47 impair host DNA degradation and phage DNA synthesis by affecting a membrane-bound DNA exonuclease. Suppressor mutations partially restore nuclease activity, indicating complex regulation of DNA metabolism during phage infection.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Lethal mutations in T4 bacteriophage genes 46 and 47 lead to incomplete host DNA degradation, premature halting of phage DNA synthesis, and defective recombination.
- These observed phenotypes suggest that genes 46 and 47 encode a DNA exonuclease, but this enzyme has not been definitively characterized in vitro.
Purpose of the Study:
- To biochemically characterize the DNA nuclease activity associated with T4 genes 46 and 47.
- To investigate the localization and properties of the proteins encoded by T4 genes 46 and 47.
- To explore the role of suppressor mutations (das) in modulating this nuclease activity.
Main Methods:
- Assaying membrane and supernatant fractions from T4-infected Escherichia coli cells for DNA degradation activity.
- Utilizing various DNA substrates (native, nicked DNA) to test nuclease specificity.
- Employing NaClO4 treatment to release proteins from membranes and DNA cellulose chromatography for protein purification and characterization.
- Investigating the effect of T4 das mutations on nuclease activity.
Main Results:
- The protein products of T4 genes 46 and 47 (gp46 and gp47) were localized to cell membranes.
- Membrane fractions from mutant-infected cells showed significantly reduced DNA degradation compared to control fractions.
- NaClO4 treatment released active gp46 and gp47, along with the associated DNase activity, which bound to DNA.
- T4 das mutations increased in vitro DNase activity in both membrane and cytoplasmic fractions.
Conclusions:
- Genes 46 and 47 of T4 bacteriophage encode a membrane-associated DNA exonuclease crucial for host DNA degradation and phage DNA replication.
- The activity of this exonuclease is regulated, as evidenced by the effects of suppressor mutations.
- This study provides the first in vitro evidence for the DNA exonuclease activity of T4 genes 46 and 47.