Nucleases endonucleases

A second explanation of the ability of oxidative stress to cause DNA damage is that the stress tri ers a series of metabolic events within the cell that lead to activation of nuclease enzymes, which cleave the DNA backbone. Oxidative stress causes rises in intracellular free Ca, which can fiagment DNA by activating Ca -dependent endonucleases (Orrenius etal., 1989 Farber, 1990 Ueda and Shah, 1992) in a mechanism with some of the features of apoptosis (see Wyllie, 1980). An example of apoptosis is the killing of immature thymocytes by glucocorticoid hormones, which activate a cell-destructive process that apparently involves DNA fragmentation by a Ca -dependent nuclease. 

Restriction endonucleases are at the core of recombinant DNA technology. The specificity of some of these enzymes is described in Chapter 13. Fortunately the restriction endo-nucleases cleave DNA at... 

Nucleases that hydrolyse phosphodiester bonds, within the nucleic acid chain are endonucleases (Figure 10.8). Apart from specificity for DNA or RNA shown by some, nucleases, these enzymes show specificity for ... 

Phosphodiester links within the chain (endonucleases) or at the end of a chain (exonucleases) for details of nucleases reactions see Appendix 10.1. 

To explain the enzymology of DNA replication, we first introduce the enzymes that degrade DNA rather than synthesize it. These enzymes are known as nucleases, or DNases if they are specific for DNA rather than RNA. Every cell contains several different nucleases, belonging to two broad classes exonucleases and endonucleases. Exonucleases degrade nucleic acids from one... 

Terms in bold are defined template 950 semiconservative replication 950 replication fork 951 origin 952 Okazaki fragments 952 leading strand 952 lagging strand 952 nucleases 952 exonuclease 952 endonuclease 952 DNA polymerase I 952 primer 954 primer terminus 954... 

A multitude of nucleases cleave DNA, single- or double-stranded. They range from the pancreatic digestive enzyme DNase I through specialized nucleases that function during DNA repair and the hundreds of restriction endonucleases that have become so valuable in modern laboratory work. Some nucleases leave a 3 -phosphate ester at a cut end in a DNA chain, while others leave a 5 -phosphate end.824 Many nucleases are dealt with in later chapters. Only a few will be mentioned here. 

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... 

Why do cells ever splice proteins It isn t clear. However, a curious fact is that many inteins are homing endonucleases.h k The genes for these nucleases are often present in introns in mRNA, and the homing endonuclease often cuts DNA in such a way as to initiate movement of its own gene (Chapter 27). The endonuclease itself is found in the center of the intein between the two end domains, which contain the catalytic centers for the splicing reaction. 

Restriction endonucleases require the presence of Mg2+ for activity. The quench buffer contains EDTA, which complexes transition metal ions in the solution. The metal ions are no longer available for binding by the nuclease molecules and enzyme activity is inhibited. 

During the past three years endonucleases have been discovered which possess a specificity which is considerably more refined than that shown by the nucleases considered thus far. Typically, these enzymes catalyze the cleavage of one or, at most, a few phosphodiester bonds in a DNA molecule composed of many thousands of nucleotide residues. In no instance has the basis for this remarkable specificity been established. However, in the case of the E. coli restriction enzymes the presence or absence of a methyl group on a specific deoxyadenylate or deoxycyti-dylate residue may be involved. 

Among endonucleases which hydrolyze DNA one seldom finds an enzyme that attacks double-stranded and single-stranded substrates with equal ease. If the enzyme shows preference for double-stranded substrates (as DNase I does) autoretardation is observed. This decrease in the reaction rate is caused by the gradual disappearance of the preferred, double-stranded substrate and an increase in the concentration of less susceptible, single-stranded substrate. Differences in rates between the early and terminal phases of the reaction of the order of 1000-fold have been described (< ). The opposite case, autoacceleration, is seen with those enzymes that show preference for the single-stranded structure, e.g., micrococcal nuclease (7). 

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