Degradation Of Duplex DNA Research Articles

Flap endonuclease of bacteriophage T7: Possible roles in RNA primer removal, recombination and host DNA breakdown.

Gene 6 protein of bacteriophage T7 has 5′-3′-exonuclease activity specific for duplex DNA. We have found that gene 6 protein also has flap endonuclease activity. The flap endonuclease activity is considerably weaker than the exonuclease activity. Unlike the human homolog of gene 6 protein, the flap endonuclease activity of gene 6 protein is dependent on the length of the 5′-flap. This dependency of activity on the length of the 5′-flap may result from the structured helical gateway region of gene 6 protein which differs from that of human flap endonuclease 1. The flap endonuclease activity provides a mechanism by which RNA-terminated Okazaki fragments, displaced by the lagging strand DNA polymerase, are processed. 3′-extensions generated during degradation of duplex DNA by the exonuclease activity of gene 6 protein are inhibitory to further degradation of the 5′-terminus by the exonuclease activity of gene 6 protein. The single-stranded DNA binding protein of T7 overcomes this inhibition.

CTG Repeats Show Bimodal Amplification in E. coli

Trinucleotide repeats in human genetic disorders showing anticipation follow two inheritance patterns as a function of length. Inheritance of 35–50 repeats show incremental changes, while tracts greater than 80 repeats show large saltatory expansions. We describe a bacterial system that recapitulates this striking bimodal pattern of CTG amplification. Incremental expansions predominate in CTG tracts < Okazaki fragment size, while saltatory expansions increase in repeat tracts ≥ Okazaki fragment size. CTG amplification requires loss of SbcC, a protein that modulates cleavage of single-stranded DNA and degradation of duplex DNA from double-strand breaks. These results suggest that noncanonical single strand–containing secondary structures in Okazaki fragments and/or double-strand breaks in repeat tracts are intermediates in CTG amplification.

Phosphatidylcholine and phosphatidylethanolamine enhance the activity of the mammalian mitochondrial endonuclease in vitro.

The purified endonuclease of bovine heart mitochondria extensively degrades a variety of DNA templates in vitro but shows a remarkably strong preference to nick within one specific evolutionarily conserved sequence block of 12 consecutive guanine residues which resides just upstream from the heavy strand origin of mitochondrial DNA replication (Low, R. L., Cummings, O. W., and King, T. C. (1987) J. Biol. Chem. 262, 16164-16170). If the enzyme serves to provide an important nicking function at this site in vivo, then mitochondrial factors likely exist which further enhance the enzyme's recognition of this locus and prevent cleavage at other less favored sites. In this study, we report that specific membrane phospholipids appear to exert such effects in vitro. In standard endonuclease assays, low levels of phosphatidylcholine or phosphatidylethanolamine (0.5 mM) stimulate the purified enzyme activity 10-20-fold. However, at moderate levels (20-40 mM), these phospholipids largely inhibit widespread degradation of duplex DNA while still allowing site-specific nicking at the conserved guanine target in the mitochondrial genome. These findings suggest that an interaction of the endonuclease with major lipid components of the inner membrane could be an important determinant of the enzyme's specificity for mitochondrial DNA.

Studies on the Mechanism of Action of the ATP‐Dependent DNAase from Alcaligenes faecalis

An ATP-dependent DNAase has been purified to homogeneity from extracts of Alcaligenes faecalis, and has been shown to couple the degradation of DNA to the hydrolysis of ATP. Enzyme activity also requires divalent ions, with Mn2+, Mg2+ and Co2+ being effective cofactors for both DNAase and ATPase activities. We have studied the intermediates formed by the enzyme during the degradation of duplex DNA with each of these cofactors using sedimentation velocity, binding to nitrocellulose filters and sensitivity to a nuclease specific for single-stranded DNA. With Mn2+ or Co2+, the enzyme acts processively to produce mostly acid-soluble material and acid-insoluble single-strand fragments up to 400-nucleotides long. However, with Mg2+ present, the enzyme produces intermediates comprising a duplex region with one or more single-strand tails, while little acid-soluble oligonucleotide is formed. From these results, we propose a model to describe the mechanism by which the ATP-dependent DNAase from A. faecalis degrades duplex DNA.

Mechanism of DNA degradation by the ATP-dependent DNase from Hemophilus influenzae Rd.

The rate of production of acid-soluble material during degradation of duplex DNA by Hemophilus influenzae ATP-dependent DNAse (Hind exonuclease V) has been shown to be directly dependent upon the Mg2+ concentration in the reaction mixture. At high concentrations of Mg2+ (5 to 20 mM), DNA degradation to acid-soluble products is rapid and the rate of ATP hydrolysis is slightly depressed. At low concentrations of Mg2+ (0.1 to 0.5 mM), the enzyme rapidly hydrolyzes ATP and converts up to 35% of linear duplex DNA to single-stranded material while degrading less than 0.2% of the DNA to acid-soluble products. We refer to this enzymatic production of single-stranded DNA as the "melting" activity. Under the conditions of our assay, the initial melting reaction is processive, lasting about 70s on phage T7 DNA. Using DNAs with several different lengths, we have established that the duration of the initial reaction is dependent upon DNA length, requiring approximately 1 s per 0.18 mum. The products of the initial reaction on phage T7 DNA are somewhat heterogeneous, consisting of short duplex fragments approximately 0.5 mum long, purely single-stranded products up to 7 mum long, and longer duplex fragments 3 to 11 mum in length, some of which have single-stranded tails. Nearly half of the single-stranded material remains linked to a duplex segment of DNA after the inital processive reaction. We propose that Hind exo V initiates attack at the DNA termini and then acts in a processive manner, migrating along the DNA molecule, converting some regions to single-stranded material by the combined action of the melting activity and limited phosphodiester cleavage, while leaving other regions double-stranded. At the completion of its processive movement through a single DNA molecule, it is released and then recycles onto either intact molecules or the partially degraded products, continuing in this manner until the DNA is finally reduced to oligonucleotides.

The degradation of duplex DNA by the recBC DNase of Escherichia coli.

The catalytic reactions of the recBC enzyme of Escherichia coli with various substrates are reviewed, and a model for the sequence of events in the degradation of duplex DNA is presented. The potential of the enzyme to take part in genetic recombination and repair is discussed.

Degradation of duplex DNA by S 1 nuclease from Aspergillus

Summary A nuclease from Aspergillus which selectively degrades single-stranded polynucleotides, S 1 , has been purified by DEAE-cellulose and Sephadex G-100 chromatography. The purified enzyme is still capable of degrading both OX174 RF and native calf thymus DNA. The hydrolysis of double-stranded DNA appears to be endonucleolytic as is single-strand cleavage.