Nuclease enzyme inhibition

Deoxyribonucleic acid footprinting studies have shown that HMG domains A and B inhibit cleavage by nucleases over a 12- to 15-base-pair region centered around the platinum adduct (81). The HMG proteins can modulate cisplatin cytotoxicity by inhibition of the excinuclease-mediated removal of Pt-d(GpG) adducts from DNA (82). However, this hypothesis has been questioned because there is no evidence for cellular protein shielding of Pt-d(GpG) adducts from repair enzymes (83). 

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. 

From differences between the spectral (29) and fluorescence (SO) properties of the inhibited and uninhibited nuclease and from differences in the number of groups susceptible to acetylation (32) in the two forms of the enzyme, Cuatrecasas et al. (S3) concluded that tyrosyl residues were involved in binding pdTp. This led them to some very interesting studies of the specific modification of certain tyrosyl residues with tetranitromethane and of the properties of these modified forms of the nuclease which we will discuss below in the context of this paper. Briefly, the pattern of relative reactivity was found to be... 

To isolate a functional macromolecular component from bacterial cells, you must accomplish three things. First, you must efficiently disrupt the bacterial cell wall and cell-membrane system to facilitate extraction of desired components. Second, you must work under conditions that either inhibit or destroy the many degradative enzymes (nucleases, proteases) released during cell disruption. Finally, you must employ a fractionation procedure that separates the desired macromolecule from other cellular components in satisfactory yield and purity. 

The isolation of bacterial DNA described in this experiment, patterned after the work of Marmur (1961), accomplishes these objectives. Bacterial cells are disrupted by initial treatment with the enzyme, egg-white lysozyme, which hydrolyzes the peptidoglycan that makes up the structural skeleton of the bacterial cell wall. The resultant cell walls are unable to withstand osmotic shock. Thus, the bacteria lyse in the hypotonic environment. The detergent, sodium dodecyl sulfate, (SDS, sodium do-decyl sulfate) then completes lysis by disrupting residual bacterial membranes. SDS also reduces harmful enzymatic activities (nucleases) by its ability to denature proteins. The chelating agents, citrate and EDTA (ethylenediamine tetraacetic acid), also inhibit nucleases by removing divalent cations required for nuclease activity. 

Since metal coordination or immobilization of the transferred phosphoryl group by multiple hydrogen bonds would inhibit the formation of a metaphosphate intermediate in an S l mechanism and would facilitate nucleophilic attack in an Sy2 mechanism, the latter process seems likely for the reactions catalyzed by staphylococcal nuclease, DNA polymerase, pyruvate kinase, fructose diphosphatase, phosphoglucomutase, (Na + K) ATPase and possibly PEP carboxylase. In creatine kinase where an S l mechanism is possible, the enzyme would have to prevent access of nucleophiles other than ADP and creatine to the reactive metaphosphate intermediate. 

Phosphorothioates interact with nucleases and DMA polymerases. These compounds are slovdy metabolized by both endo- and exonucleases and inhibit these enzymes (160, 171). The inhibition of these enzymes appears to be competitive and this may account for seme early data suggesting that phosphorothioates are almost infinitely stable to nucleases. In these studies, the oligonucleotide-to-enzyme ratio was very high and thus the enzyme was inhibited. Phosphorothioates also bind to RNase H when in an RNA-DNA duplex... 

One mechanism intrinsic to virtually aU DNA polymerases is a separate 3 5 exonuclease activity that double-checks each nucleotide after it is added. This nuclease activity permits the enzyme to remove a newly added nucleotide and is highly specific for mismatched base pairs (Fig. 25-7). If the polymerase has added the wrong nucleotide, translocation of the enzyme to the position where the next nucleotide is to be added is inhibited. This kinetic pause provides the opportunity for a correction. The 3 5 exonuclease activity removes the mispaired nucleotide, and the polymerase begins again. This activity, known as proofreading, is not simply the reverse of the polymerization reaction (Eqn... 

The nuclease which degrades oligo-isoadenylate copurified with the iso-A activated ribonuclease P over several steps, but was separated on hydroxylapatite. The iso-A degrading enzyme (nuclease D of Figure 1) splits (2 -5 ) P-A- to yield 5 -AMP, and more slowly (3 -5 )-A-pA. It clearly differs from known ribonucleases that are inhibited by 2 -5 dinucleotides (22). 

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