Incision at Nucleotide Insertions/Deletions and Base Pair Mismatches by the SP Nuclease of Spinach

• Published in: Biochemistry (Easton) Vol. 38; no. 7; pp. 2200 - 2205
• Main Authors: Oleykowski, Catherine A, Bronson Mullins, Colleen R, Chang, David W, Yeung, Anthony T
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 16-02-1999
• Subjects: Adenine - chemistry; Base Pair Mismatch; Base Sequence; DNA Repair; Endonucleases - chemistry; FEUILLE; HOJAS; Hydrogen-Ion Concentration; LEAVES; Molecular Sequence Data; NUCLEASAS; NUCLEASE; NUCLEASES; Oligonucleotides - chemistry; Osmolar Concentration; Plant Proteins - chemistry; Sequence Deletion; SPINACIA OLERACEA; Spinacia oleracea - enzymology; Substrate Specificity; Thymine - chemistry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi982318y

Spinach leaves contain a highly active nuclease called SP. The purified enzyme incises single-stranded DNA, RNA, and double-stranded DNA that has been destabilized by A-T-rich regions and DNA lesions [Strickland et al. (1991) Biochemistry 30, 9749−9756]. This broad range of activity has suggested that SP may be similar to a family of nucleases represented by S1, P1, and the mung bean nuclease. However, unlike these single-stranded nucleases that require acidic pH and low ionic strength conditions, SP has a neutral pH optimum and is active over a wide range of salt concentrations. We have extended these findings and showed that an outstanding substrate for SP is a mismatched DNA duplex. For base-substitution mismatches, SP incises at all mismatches except those containing a guanine residue. SP also cuts at insertion/deletions of one or more nucleotides. Where the extrahelical DNA loop contains one nucleotide, the preference of extrahelical nucleotide is A ≫ T ∼ C but undetectable at G. The inability of SP to cut at guanine residues and the favoring of A-T-rich regions distinguish SP from the CEL I family of neutral pH mismatch endonucleases recently discovered in celery and other plants [Oleykowski et al. (1998) Nucleic Acids Res. 26, 4597−4602]. SP, like CEL I, does not turn over after incision at a mismatched site in vitro. Similar to CEL I, the presence of a DNA polymerase or a DNA ligase allows SP to turn over and stimulate its activity in vitro by about 20-fold. The possibility that the SP nuclease may be a natural variant of the CEL I family of mismatch endonucleases is discussed.