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Provides key information on a wide range of enzymes commonly used as tools in molecular biology, helping to minimize the time a scientist spends researching the literature to get reactions to work efficiently and allowing the nonenzymologist to design an experiment. Each chapter gives background information on the enzyme selected and those parameters important in its use, describes both the source and application of the enzyme, and provides details on the size and structure of the protein. Specific parameters essential for achieving an optimized reaction are discussed, along with exemplary practical procedures an protocols.
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CHAPTER1
Nucleases An Overview
A. Fred Weir Enzymes able to digest nucleic acids are of course essential to molecular biology, indeed the whole technology was founded on the discovery of bacterial enzymes that cleave DNA molecules in a base-specific manner. These enzymes, the type II restriction endonucleases, are perhaps the best studied of the nucleases as to both their in vivo role and their use as tools in the techniques of molecular biology. However, the nucleases are ubiquitous in living organisms and function in all situations where partial or complete digestion of nucleic acid is required. These situations not only include degradation and senescencebut also replication and recombination, although it must be noted that, to date, evidence for the involvement of nucleases in the latter two processes in eukaryotes is largely circumstantial. The significance of nucleases in the functioning of nucleic acids as the genetic material can be gaged however by considering that several enzymes implicated in DNA replication, recombination, and repair have integral exo- or endodeoxyribonuclease activity. For example, the 5’-3’ and 3’-5’ exonuclease activity of DNA polymerases and the endo-DNase activity of topoisomerases (e.g., see ref. I). As well as the restriction endonucleases, various other nuclease enzymes have been used as tools in molecular biology, the purpose of this chapter is to give some background on the main deoxyribonu-
From Methods In Molecular Biology, Vol 16 EnZym8S of Molecular Biology Edited by: M. M Burr811 Copynght 01993 Humana Press Inc , Totowa, NJ
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cleases (DNases) and then to focus on the techniques in which they are used. The enzymes that molecular biologists use as tools are dealt with in separate chapters in this volume. 1.1. Nomenclature
Anyone who has tried the isolation of a DNase enzyme will know that the presence of multiple types of nuclease activity makes this process fraught with difficulty. In this section, consideration will be given to the properties of the DNase enzymes with a view to understanding their nomenclature, which for the most part is somewhat confusing (Table 1). Nucleases, although a large group in themselves, are part of a larger group of enzymes, the phosphodiesterases, which are able to catalyze the cleavage of phosphate-esterbonds. Schmidt and Laskowsi (2) identified three types of nuclease enzymatic activity: DNases, ribonucleases (RNases), and exonucleases. On this definition, it is apparent that so-called DNases and RNasescleave their substratesendonucleolytically, i.e., at internal sites, and that this activity is distinct and separable from any exonuclease activity. In practical terms, this definition holds true in that an endo-DNase will not digest DNA molecules to completion, i.e., to nucleotide monomers; only when exonuclease activity is present will the digestion of DNA go to completion, A second confusing element in the nomenclature of nucleases, and DNases in particular, is the presence of single-stranded DNases, e.g., mung bean nuclease and nuclease S1 from Aspergillus. These enzymes, although having high specificity for single-stranded DNA molecules, will