Enzyme assay endonuclease

Single-cell alkaline gel electrophoresis (the Comet assay) is a sensitive assay of DNA strand breaks. The Comet assay can be adapted to measure oxidative damage to DNA bases by measuring strand breaks induced by the treatment of DNA with relevant repair enzymes, eg., endonuclease III (EndoIII) for oxidized pyrimidines and formamidopyrimidine glycosylase (Fpg) for 8-oxogua and ring-opened pyrimidines, such as those resulting from the breakdown of alkylated bases. ... 

Utilising a reversion assay in Salmonella enterica, Prieto et al reported an increased frequency of point mutations following bile-salt exposure. Mutations were predominantly nucleotide substitutions (GC to AT transitions) and -1 frameshift mutations.The frameshifts were dependent on SOS induction and linked to the activity of DinB polymerase (Pol IV). The authors proposed that the GC to AT transitions stimulated by bile, could have arisen from oxidative processes giving rise to oxidised cytosine residues. Consistent with this hypothesis, the authors demonstrated that strains of S. enterica-lacking enzymes required for base-excision repair (endonuclease III and exonuclease IV) and the removal of oxidised bases, demonstrated increased bile-acid sensitivity compared with competent strains. In another study using E. coli, resistance to the DNA-damaging effects of bile was associated with Dam-directed mismatch repair, a pathway also involved with the repair of oxidative DNA lesions. ... 

The role of endonuclease II in vivo is not known. Assay of recombination defective mutants of E. coli and mutants abnormally sensitive to ultraviolet irradiation and to treatment with methyl methane sulfonate showed them all to possess normal levels of the enzyme (56). 

The application of directed evolution approaches for the change or extension of the specificity of a restriction enzyme is hampered by the fact that an in vivo selection assay is not available and that examination of endonuclease activity in vitro usually requires purification of the enzymes to avoid background activity by other nucleases prevalent in all cells. This means that an altered or extended specificity can only be observed with sufficiently purified protein preparations, thereby unfortunately separating genotype and phenotype. As neither a reliable in vivo test nor the secretion of restriction endo-... 

Restriction-site mapping in DNA may be performed by end-labelling with polynucleotide kinase as above, and subsequent partial digestion with the restriction enzyme. An overlapping series of polynucleotides with a common labelled terminus is thus formed, and size analysis of these affords a restriction map. Polynucleotide kinase may also be used to assay the number of 5 -phosphorylated termini in DNA generated as the result of the action of restriction endonuclease. In the presence of... 

DNA pol a with primase was assayed in a reaction mixture (0.2 ml) containing 50 mM Tris-HQ buffer, pH 7.4, 10 mM MgCU, 1 mM ATP, 2.5 Mg of poly(dT), 20mM[ H]-dATP (35 cpm pmoF ) and an appropriate amount of the enzyme. The reaction was carried out at 37°C for 1 h. DNA polymerase a and i3 activity was assayed with activated DNA as template-primer. Details of the assay will be described elsewhere [30]. The assay of DNA ligase [12,13], Ca ", Mg -dependent endonuclease [14], and terminal deoxynucleotidyl transferase [15] was carried out according to the respective reported method. 

The cis-syn thymine photodimer can be repaired by the nucleotide excision repair (NER) pathway by endonucleases such as XPF. Using a yeast two-hybrid assay it has been shown that XPF interacts with the p70 subunit of the single-stranded DNA binding protein RPA in the process of excision of CPD at the 5 -thymine. Another key class of enzyme involved... 

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