A novel N-glycosylated, mannose-rich protein has been purified approx. 4000-fold from human liver in a seven-step procedure including ion-exchange chromatography and fractionation on concanavalin A-Sepharose, Sephadex G-75 and oligo(dT)-cellulose matrices. The molecular mass of the protein is 46 kDa when measured by gel filtration (i.e. under non-denaturing conditions) and 60 kDa by SDS/PAGE (i.e. under denaturing conditions). The protein possesses two DNA backbone-incising activities, namely, the random introduction of single-strand breaks in native DNA and the rupture of the phosphodiester linkage internal to cyclobutyl pyrimidine dimers, the major class of DNA lesions induced by solar UV rays. Both activities are optimal at pH 5.0 in vitro, although the non-specific nuclease displays appreciable activity at neutral pH, depending on the buffer composition. The protein has been named acidic nuclease/intra-cyclobutyl-pyrimidine-dimer-DNA phosphodiesterase (AN/IDP). As a nuclease, the protein ‘prefers#x2019; a linear DNA structure over a covalently closed circular molecule and is more proficient at digesting single-stranded than double-stranded DNA. The polynucleotide cleavage products of the nuclease contain 5ʹ-OH and 3ʹ-PO4 termini, which are refractory to direct rejoining by DNA ligases. Depending on the substrate, the nuclease activity exhibits a temperature optimum of 50 °C or greater, and is neither stimulated by Mg2+ or Ca2+ nor inhibited by Zn2+. AN/IDP is present in human liver and in cultured human cells of both fibroblastic and lymphocytic origins. Intracellularly, the protein can be readily detected in both the cytosolic and nuclear fractions, although much more (approx. 3-fold) is found in the latter fraction. We propose that this bifunctional enzyme may be involved in both apoptotic DNA digestion and metabolism of cyclobutyl pyrimidine dimers in UV-irradiated human cells.
Purification and characterization of a novel human acidic nuclease/intra-cyclobutyl-pyrimidine-dimer-DNA phosphodiesterase
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Konrad S. FAMULSKI, Michel LIUZZI, Saber BASHIR, Razmik MIRZAYANS, Malcolm C. PATERSON; Purification and characterization of a novel human acidic nuclease/intra-cyclobutyl-pyrimidine-dimer-DNA phosphodiesterase. Biochem J 1 February 2000; 345 (3): 583–593. doi: https://doi.org/10.1042/bj3450583
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