Characterization Of Endonuclease Activity Research Articles

Characterization of endonuclease activity from excretory/secretory products of a parasitic nematode, Trichinella spiralis.

Double-stranded endonuclease activity was demonstrated for the first time in the excretory/secretory (ES) products of a parasitic nematode, Trichinella spiralis, which can reorganize host muscle cells. The endonuclease introduced double-stranded breaks to the native DNA. The ES double-stranded endonuclease(s) was sequence nonspecific, with a pH optimum below 6, and required divalent cations as a cofactor. Its activity was inhibited by the Zn2+ ion. It was detected mainly in the ES products of the infective-stage larvae of T. spiralis collected at 37 degrees C and was present in much smaller amounts in samples collected at 43 degrees C and in the products of T. pseudospiralis, a nonencapsulated species. The activity of endonuclease was blocked by antibodies against ES products. Zymographic analysis showed that the endonuclease activity was associated with at least three molecular forms, designated approximately 25, 30 and 58 kDa, respectively.

Characterization of an endonuclease activity which preferentially cleaves the G-rich immunoglobulin switch repeat sequences

B lymphocytes can alter selectively their immunoglobulin (Ig) isotype expression by deletional rearrangement of the first active immunoglobulin heavy-chain (IgH) constant region (Cμ) gene with one of six other constant region genes. Recombination breakpoints occur within highly repetitive “switch” (S) regions located upstream of each IgH constant region gene except C δ. Analysis of rearranged switch DNA junctions has not detected a consensus sequence, although the predominance of two pentamer motifs (TGGGG and TGAGC) at or near these breakpoints and throughout all murine S region sequences has led to their advocacy as the S recombination signals. In this paper, we describe the characterization and partial purification of a lymphoid-specific endonuclease activity which cleaves preferentially murine S region DNA. Enzyme activity selectively produced single- and double-stranded breaks at TGAGC and TGGG motifs within murine Sμ and Sα DNA. Rare cryptic cleavage sites were detected also within non-switch sequences, although cleavage intensities at these sites were reduced greatly, relative to consensus S region cleavages. Analogous activity was found in murine tissue extracts, although among the tissues assayed only spleen and thymus contained detectable activity. Subsequent biochemical characterization of this activity demonstrated that the responsible enzyme (Endo-SR) represented a previously unreported tissue-specific mammalian endonuclease. Endo-SR-specific activity could be enhanced by addition of Mg 2+ or Ca 2+ and inhibited by addition of Zn 2+. Maximal specific activity was detected at pH 5.5 and sharply declined within ±0.5 pH units. In view of this enzyme's sequence- and tissue-specificity, we propose that Endo-SR is a strong candidate for an endonuclease activity associated with the switch recombination process.