Enzymes of Molecular Biology Chapter 1
Author: Eze-Odikwa Tochukwu Jed
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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 senescence but 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 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 deoxyribonucleases (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.
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-ester bonds. 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 RNases cleave their substrates endonucleolytically, 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 S 1 from Aspergillus. These enzymes, although having high specificity for single-stranded DNA molecules, will, at high concentrations and in preparations not purified to homogeneity, digest native (double-stranded) DNA molecules albeit at reduced rates. For an example of this, Weir and Bryant (3) have isolated a nuclearlocated DNase from the embryo axes of pea that has a low, but measurable activity on native DNA but rapidly catalyzes the hydrolysis of heat-denatured DNA. It is not known so far whether these two activities are separable, but evidence from similar enzymes suggests that these activities are part of the same protein molecule. DNases then, tend to be classified as to “what they do best”; e.g., the DNase of Weir and Bryant would be called a single-strand specific endo-DNase. In the following discussion, the examples are from the DNase class of nucleases, however all the points considered can be equally applied to the RNases.
Table 1 The Nomenclature of Nuclease
|3.1. I 1||Exodeoxyribonucleases producing 5′ phosphomonoesters.||Exonuclease III |
|3.1.1||Exoribonucleases producing 5′-phosphomonoesters.||Exo-RNase H|
|3.1.15||Exonucleases active with either ribo- or deoxyribonucleic acids and producing 5′-phosphomonoesters.||Venom|
|3.1.1||Exonucleases active with either ribodeoxyribonucleic acids and producing other than 5′-phosphomonoesters.||Spleen|
|3.1.2||Endodeoxyribonucleases producing 5′-phosphomonoesters.||DNase I|
|3.1.22||Endodeoxyribonucleases producing other than 5′-phosphomonoesters.||DNase II|
|3.1.25||Site-specific endodeoxyribonucleases: specific for altered bases.|
|3.1.26||Endoribonucleases producing 5′-phosphomonoesters.||RNase H|
|3.1.27||Endoribonucleases producing other than 5′-phosphomonoesters.||RNase T1|
|3.1.30||Endonucleases active with either ribo- or deoxyribonucleic acids and producing 5′-phosphomonoesters.||Aspergillus|
nuclease S1 and
Mung bean nuclease
|3.1.31||Endonucleases active with either ribo- or deoxyribonucleic acids and producing other than 5′-phosphomonoesters.||Micrococcal|
1.1.1. Criteria Used for Classification
220.127.116.11. Exo- vs ENDONUCLEOLYTIC CLEAVAGE
Exo-DNases cleave from the ends of DNA molecules releasing phosphomononucleotides. Cleavage can be either in the 3′ to 5′ direction releasing 5′ phosphomononucleotides or in the 5′ to 3′ direction to yield 3′ phosphomononucleotides. An example of a widely used exonuclease is exonuclease III from Escherichia coli (EC 18.104.22.168), which will digest one strand of a double-stranded DNA molecule from a 3′ overhang or blunt end. This property has been used to produce bidirectional or unidirectional nested deletion of templates for sequencing. Endo-DNases cleave at internal phosphate bonds. Cleavage of double-stranded DNA substrates can be by a “single-hit” or a “double hit” mechanism (4) or by a combination of both (see Chapter 2, Section 2.4.). Essentially this means that the enzymes can either cleave the two strands of the DNA molecule at points opposite or at sites on the two strands that are well away from each other. The scission of the molecule will take place at a relatively faster rate in the former case as compared to the latter. The prime example of an endo-DNase is pancreatic DNase (DNase I, EC 22.214.171.124). Under optimal conditions this enzyme uses a double-hit mechanism for cleavage of substrates.
126.96.36.199. BASE SPECIFICITY AT OR NEAR THE SITE OF CLEAVAGE
None of the eukaryotic enzymes so far isolated appear to have such specificity, however there is evidence that enzymes with optimal activity on single-stranded DNAs will preferentially cleave at A-T rich sites in native DNA molecules (3,5,6). As already mentioned, the Type II restriction endonucleases have absolute specificity for a group of bases at or near the cleavage site.
188.8.131.52. SITE OF CLEAVAGE
The site of cleavage can be on either side of the phosphate bond leading to a 5′ or a 3′ monoesterified product. No enzymes have been isolated that can split the internucleotide bond on either side. This property is particularly important if the DNA molecule is to be subsequently made blunt-ended for a ligation experiment. DNA molecules leh with a 5′ overhang from a staggered cut are usually filled in with the Klenow fragment of E. coli DNA polymerase, whereas those with a 3′ overhang have the overhang cleaved back with the exonuclease activity of T4 DNA polymerase to create blunt ends.
184.108.40.206. CLEAVAGE OF NATIVE OR SINGLE-STRANDED DNA
Nuclease tend to have a “preference” for cleavage of single-stranded DNA or double-stranded DNA substrates. In general single-strand specific DNases, such as nuclease SI from Aspergillus (EC 220.127.116.11), will digest native DNA if the enzyme concentration is high. Nuclease S1 is used to analyze the structure of DNA-RNA hybrids and in cDNA synthesis where it opens the hairpin loop generated during the synthesis.
DNases have also in the past been classified according to their pH optima and requirements for the presence of metal ions; there are two major mammalian DNases–one working at neutral pH the other in acidic conditions–and both require Mg ions. Other nuclease enzymes exist that require Ca 2+ ions (pea nuclear DNase ) or Zn 2+ ions (nuclease S 1). In addition to the aforementioned properties, it is important to note that many crude preparations of DNase exhibit nonspecificity for the sugar moiety of nucleic acids, i.e., they will cleave both RNA and DNA. In these cases it is obviously essential to remove contaminating RNase activity before the enzyme is used to remove DNA from a preparation of RNA.
From the foregoing discussion, it can be seen that the nucleases arc a complex group of enzymes. However, when it comes to their use as tools in molecular biology, the situation is very much simplified as only a handful of enzymes arc used routinely in experimental protocols. The enzymes DNases I and II, exonuclease, nuclease S l, Bal3 l, and RNase will be discussed in much more detail in the following sections with special emphasis on the experimental protocols for their use in molecular biology techniques.
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