Enzymes to DNA

Ruth, J.L. (1993) Direct attachment of enzymes to DNA probes. In Methods in Nonradioactive Detection (G.C. Howard, ed.), pp. 153-177. Appleton and Lange, Norwalk, Connecticut. 

Before precise questions can be asked at the molecidar level about the important interactions of ADPRT with its substrates and with chromatin, further information is required concerning the response of the enzyme to stimuh under more nearly physiological conditions. To this end we have undertaken studies of the kinetics of ADPRT in permeabilised cells and have attempted to analyse the response of the enzyme to DNA strand breaks. 

RKamycins a group of antibiotics produced by Streptomyces medilerranei. They contain a naphthalene ring system bridged between positions 2 and 5 by an aliphatic chain. Rifamycin SV and rifampicin inhibit DNA-dependent RNA synthesis in prokaryotes, chloroplasts and mitochondna, but not in the nuclei of eukaryotes Inhibition is due to the formation of a stable complex between RNA polymerase and R. binding of the enzyme to DNA still occurs, but incorporation of the first purine nucleotide into RNA is prevented. Thus R. specifically inhibit initiation of RNA synthesis, but not chain elongation. Some R. also inhibit eukaryotic and viral RNA-poly-... 

The role played by the various subunits in the polymerase reaction has only begun to be clarified. A a factor has been isolated from E. coli which specifically stimulates the initiation of ribosomal cistron transcription. jS -Factor is believed to constitute the binding site of the core enzyme to DNA. Whether the jS -unit also contains the active site remains to be demonstrated. The (7-unit recognizes the initiation codon and facilitates specific binding. 

The quantum mechanical techniques discussed so far are typically appUed to moderate-sized molecules (up to about 100 atoms for ab-initio or DFT and up to 500 for semi-empirical MO techniques). However, what about very large systems, such as enzymes or DNA, for which we need to treat tens of thousand of atoms. There are two possible solutions to this problem, depending on the application. 

A very important issue - disregard of which is a big source of bad modeling studies - is the dear distinction of transport processes (toxicokinetics) and interactions with targets such as membranes, enzymes, or DNA (toxicodynamics). Figure 10.1-6 gives a rather simplified model of a fish to illustrate this distinction. 

Once the broad outlines of DNA replication and protein biosynthesis were established scien tists speculated about how these outlines af fected various origins of life scenarios A key question concerned the fact that proteins are re quired for the synthesis of DNA yet the synthesis of these proteins is coded for by DNA Which came first DNA or proteins How could DNA store genetic infor mation if there were no enzymes to catalyze the polymerization of its nucleotide components How could there be proteins if there were no DNA to code for them ... 

Reverse transcriptase (Section 28 13) Enzyme that catalyzes the transcription of RNA to DNA Ribozyme (Section 28 11) A polynucleotide that has catalytic activity... 

On homogenization, the lysate may drastically increase in viscosity due to DNA release. This can be ameliorated to some extent using multiple passes to reduce the viscosity. Alternatively, precipitants or nucleic acid digesting enzymes can be used to remove these viscosity-enhancing contaminants. 

Endonuclease-catalyzed hydrolysis of DNA at the internucleosomal linker regions into multimers of 180 base pairs which are visualized by electrophoresis as a ladder of nuclear DNA fragments. Access of the endonuclease to DNA is facilitated by depletion of polyamines, and the activity of the enzyme is mcrea.sed by and decreased by ADP-tibosylation. Thus, agents that increase intracellular Ca " or inhibit l>oly(ADP-ribose) polymerase can induce apoptosi.s. ... 

Reverse transcriptase (Section 28.13) Enzyme that catalyzes the transcription of RNA to DNA. 

The requirement for reduction prior to DNA alkylation and crosslinking was first demonstrated by Iyer and Szybalski in 1964 [29], and can be induced both by chemical reducing agents such as sodium dithionite and thiols in vitro and by various reductive enzymes such as DT-diaphorase (NAD(P)H-quinone oxidoreduc-tase) in vitro and in vivo [47]. Much work to characterize the mechanism of reductive activation and alkylation has been carried out, principally by the Tomasz and Kohn groups, and Figure 11.1 illustrates a generally accepted pathway for mitomycin C [16, 48-50] based on these experiments, which is very similar to the mechanism originally proposed by Iyer and Szybalski [29]. 

While many diseases have long been known to result from alterations in an individual s DNA, tools for the detection of genetic mutations have only recently become widely available. These techniques rely upon the catalytic efficiency and specificity of enzyme catalysts. For example, the polymerase chain reaction (PCR) relies upon the ability of enzymes to serve as catalytic amplifiers to analyze the DNA present in biologic and forensic samples. In the PCR technique, a thermostable DNA polymerase, directed by appropriate oligonucleotide primers, produces thousands of copies of a sample of DNA that was present initially at levels too low for direct detection. 

---