Nuclease SP: a novel enzyme from spinach that incises damaged duplex DNA preferentially at sites of adenine

Abstract

A novel endonuclease has been isolated from extracts of spinach leaves (Spinacia oleracea). The enzyme has been purified by a series of column chromatography steps and has a molecular size of approximately 43,000 daltons. The spinach endonuclease cleaved double stranded DNA damaged by ultraviolet light or cis-diamminedichloroplatinum (II) primarily at sites of adenine when end-labelled DNA fragments of defined sequence were employed as substrates. The nature of the structural distortion contained in damaged, duplex DNA appears to be an important determinant for endonuclease cleavage. DNA helical distortions produced by UV light-induced (6-4) pyrimidine-pyrimidone photoproducts, but not cyclobutane pyrimidine dimers are recognized by the enzyme. The DNA cleavage products generated by the enzyme contain 3'-hydroxyl and 5'-phosphoryl termini. Single stranded DNA and RNA are hydrolyzed by the spinach endonuclease. This enzyme, which we call nuclease SP, is similar in several respects to other single-strand-specific nucleases such as N. crassa and mung bean nucleases and may function in DNA repair and/or recombination events in spinach cells. Nuclease SP should be a useful tool for the analysis of (6-4) photoproducts occurring in duplex DNA.

Enzymatic Mutation Detection Technologies

“…On the other hand, it might be relatively easy to disable the catalytic residues of CEL I but retain the mismatch binding properties. This effort might be guided by studying the Spinach nuclease (SP; References 43, 52, and 53), which shows properties intermediate between CEL I and S1 (43). For example, highly purified SP is able to cut mismatches, but not those with guanine bases in the mismatch site.…”

“…The latter is a crucial factor in high-throughput basic research projects but not recommended for the occasional users or for commercial or clinical applications. New methods for the detection of known mutations at high sensitivity continue to be invented (53,54). However, for the detection of unknown mutations, the CEL I mismatch detection approach provides the simplest yet flexible method in our opinion, for both short-term and long-term mutation detection projects.…”

Single-strand-specific nucleases

“…Their presence has also been shown in the endoplasmic reticulum, Golgi apparatus, protein bodies and vacuoles of the aleurone layer of barley seeds [38], bound to chromatin in the embryo axis of germinating pea [39], cultured tobacco cells [40] and leaves of Avena [41], spinach [42] and tea [43]. Moreover, they have also been isolated from mung bean sprouts [44], germinating pea seeds [45] and barley [46], potato tubers [47] and tobacco pollen [48].…”

“…This suggested that the enzyme does not cleave DNA in response to speci¢c adenine modi¢cation but rather incises DNA at adenine residues in the vicinity of helical distortions produced by photoproducts, platinum and other modi¢ca-tions. Hence, it was concluded that SP nuclease might be involved in the repair of the dimers formed due to UVirradiation [42]. Enzymatic hydrolysis of the intradimer phosphodiester bond may constitute the initial step in the repair of UV-induced cyclobutane pyrimidine dimers in human cells.…”

Molecular cloning and characterization of a cDNA encoding endonuclease from potato (Solanum tuberosum)

“…Sss nucleases have also been isolated from various cellular components, and derived from the tissues or organs of many plant species (reviewed by Desai and Shankar, 2003), for instance, nucleases from mung bean (Sung and Laskowski, 1962;Laskowski, 1980), spinach (Doetsch et al, 1988) and, of special interest in relation to this paper, from potato (Nomura et al, 1971). More recently, three different nuclease I cDNAs have been isolated from Zinnia (Aoyagi et al, 1998;Perez-Amador et al, 2000).…”