Endonuclease nonspecific

Viadiu, H., Kucera, R., Schildkraut, 1. and Aggarwal, A. K. (2000). Crystallization of restriction endonuclease BamHI with nonspecific DNA. J. Struc. Biol. 130, 81-85. 

Attempts are being made to design semisynthetic restriction endonucleases specific for single-stranded DNA or RNA. For example, an oligonucleotide with a sequence complementary to a sequence adjacent the linkage that is to be cut can be covalently linked to a relatively nonspecific nuclease. Such an enzyme derived from micrococal nuclease cuts a single-stranded chain of either DNA or RNA adjacent to the double-stranded region of the ES complex.839... 

Adenosine 2, 3 -cyclic phosphate is scarcely accumulated, though other nucleoside 2, 3 -cyclic phosphates are accumulated as intermediates. This result suggests that the action of RNase T2 on RNA is owing to the cooperation of an adenylic acid specific endonuclease activity and a nonspecific exonuclease activity releasing mononucleotides from the 3 terminal. 

First, exonuclease (phosphodiesterase), endonuclease (8, 10-12), 5 -nucleotidase (13), and nonspecific phosphatase (13) are present in all venoms that have been analyzed. Therefore, nature did not provide these materials as sources of any one of these enzymes free from undesirable contaminants which may be represented by the others. 

Several methods for preparation of purified venom exonuclease have been described [see the reviews on methods (15, 18, 19, 21) and books (22-24)]. The major effort of purification was directed toward removing the contaminating monophosphatases. A successful and widely used step was introduced by Sinsheimer and Koerner (43). At pH 4, monophosphatases are precipitated with a lower concentration of acetone the remaining exonuclease is precipitated by a higher acetone concentration. Several modifications of this principle have been proposed (concentration of acetone, temperature during precipitation, etc.). Commercially available preparations represent essentially this stage and contain per unit of exonuclease 10 4 unit of 5 -nucleotidase, 10 3 unit of nonspecific phosphatase, and about the same amount of endonuclease. The last figure is only an approximation because of the difficulty of accurate determination. The more elaborate preparations have these contaminants... 

Fig. 5. Schematic of the construction and function of chimeric restriction endonucleases (Chandrasegaran and Smith, 1999). (a) The type IIS restriction enzyme Fokl has a nonspecific endonuclease domain (FN) and a DNA-binding domain. Swapping Fokl s DNA binding-domain for that of another DNA binding-domain results in a chimeric restriction enzyme with new specificity. The flexibility resulting from less intimate contact between the Fn domain and its new DNA-binding domain translates into DNA cleavage at several locations near the binding site, (b) The modular nature and tunable specificity of zinc...

Fig. 3. Universal primers for PCR amplification of endogenous retroviral reverse transcriptase. The primers anneal within the retroviral reverse transcriptase gene at the position indicated by the thick lines in Fig. 2. The degenerate primer pair has been used successfully,21 but it gives a number of nonspecific products. Substitution of inosine at some of the degenerate positions increases the effective concentration of primer. Xba and coRI restriction endonuclease cleavage sites are underlined.

These enzymes are extraordinarily abundant over 1200 restriction endonucleases had been isolated and characterized by early 1990. Of three classes defined, type II restriction enzymes, which generally cut within their recognition sequences, have found uses in a host of biomedical research and diagnostic applications to be discussed below. Type 1 enzymes cut nonspecifically many nucleotides distal to specific recognition sequences and contain both restriction enzyme and DNA modification (see below) activities on different subunits of multienzyme complexes. Type III restriction enzymes share the multienzyme aspeas of type I enzymes but vary in other properties such as ATPase activity and cofactor requirements. 

Figure 9.41. Nonspecific and Cognate DNA within ECOViX Endonuclease. A comparison of the positions of the... 

Figure 9A1 Nonspecific and cognate DNA within coRV endonuclease, A comparison of the positions of the nonspecific (orange) and the cognate DNA (red) within JEcoRV. Notice that, in the nonspecific complex, the DNA backbone is too far from the enzyme to complete the magnesium ion-binding sites. [Drawn from IRVB.pdb.]...

Nonspecific and cognate DNA within EcnR r endonuclease Figure 9.41... 

The answer is a. (Murray, pp 435-451. Scriver, pp 3-45. Sack, pp 1-40. Wilson, pp 101-120.) In mammals, RNA polymerase binds to promoter sites upstream from the start site. These include the TATA box (TATAAT), the CAAT box, and the GC box. DNA primase and helicase are involved in DNA replication and do not bind specifically to sequences upstream of genes. Restriction endonucleases recognize specific sequences in double-helical DNA and cleave both strands. Histones nonspecifically bind to chromosomal DNA and constitute about half the mass of mammalian chromosomes. 

If naked DNA (that is, DNA that is not protein-complexed) is partially digested with a nonspecific endonuclease that randomly cuts double strands, a broad smear of polynucleotide fragments is observed in an electrophoresis gel. If the same experiment is conducted with chromatin, or even with whole nuclei (which the nuclease can easily penetrate through the nuclear pores), the random DNA cleavage yields a series of bands that are multiples of approximately 200 base pairs. This indicates that "naked" DNA is present only at regularly spaced points. 

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