Deoxyribonuclease I
I.U.B.: 3.1.21.1
Deoxyribonucleate 5'-oligonucleotidohydrolase
Deoxyribonuclease from beef pancreas, DNase I, first crystallized by Kunitz (1950), is an endonuclease, splitting phosphodiester linkages, preferentially adjacent to a pyrimidine nucleotide yielding 5'-phosphate terminated polynucleotides with a free hydroxyl group on position 3'. The average chain of limit digest is a tetranucleotide (Matsuda and Ogoshi 1966). DNase I acts upon single chain DNA (Young and Sinsheimer 1965), and upon double-stranded DNA and chromatin. In the latter case, although histones restrict susceptibility to nuclease action, over a period of time nearly all chromatin DNA is acted upon. According to Musky and Silverman (1972), this could result from the looseness of histone attachment to DNA. They found that lysine-rich histones more effectively block DNase access to DNA than arginine-rich histones. Billing and Bonner (1972) suggest that DNase attacks the histone-free strand of chromatin DNA. Schmidt et al. (1972) indicate that hydrolysis of the histone-free region of DNA strands accounts for the initial rapid action of the enzyme on chromatin. Bollum (1965) reports degradation of synthetic homopolymer complexes by DNase I. The intracellular functions of the enzyme are probably controlled by a DNase inhibitor (Lindberg and Skoog 1970), which according to Lazarides and Lindberg (1974) is actin.
Characteristics of the Enzyme from Bovine Pancreas:
Molecular weight: 31,000 (Lindberg 1967).
Composition: There are four deoxyribonucleases of beef pancreas: A, B, C, and D (Salnikow, Moore, and Stein 1970; Salnikow and Murphy 1973; Liao 1974). Five have been reported by Junowicz and Spencer (1973a). They are glycoproteins differing from each other either in carbohydrate side-chain or polypeptide component. (Liao 1974; Salnikow and Murphy 1973.) DNase A is the predominant form (Salnikow et al. 1973a); its amino acid sequence has been reported (Liao et al. 1973).
Optimum pH: 7.8.
Extinction coefficient: = 11.1.
Activators: DNase I is activated by bivalent metals (Junowicz and Spencer 1973; Poulos and Price 1972; Price 1972; Price 1975). Maximum activation is attained with Mg2+ plus Ca2+. It has been indicated that a metallosubstrate, such as Mg salt of DNA might be necessary (Perlgut and Hernandez 1972; Poulos and Price 1972; Douvas and Price 1975; Price 1975).
Specificity: See Clark and Eichhorn (1974), and Bernardi et al. (1975).
Inhibitors: According to Davidson (1972), citrate completely inhibits magnesium-activated but not manganese-activated enzyme. DNase I is inhibited by chelating agents such EDTA (Junowicz and Spencer 1973), and sodium dodecyl sulfate (Liao 1975a).
Stabilizers: The most likely proteolytic contaminant of DNase I is chymotrypsin B. Price et al. (1969) report that DNase I can be stabilized against proteolytic digestion by 5 mM Ca2+. Diisopropylfluorophosphate (DFP) may also be used to inhibit contaminating proteases (Laskowski 1966).
Stability: The lyophilized enzyme is stable for 2-5 years when stored at 5°C.