Polynucleotide ligase from myeloid and lymphoid tissues.

Abstract

Enzymes with the property of repairing single-strand interruptions in doublestranded DNA molecules are presumed to be important in recombination, radiation repair, and DNA replication. Two such enzymes, designated polynucleotide ligases, have recently been isolated from bacteria. Both enzymes catalyze the formation of a 3'-5'-phosphodiester bond, starting with a 5'-phosphoryl terminus and a 3'-hydroxyterminus, juxtaposed at a single-strand break within double-stranded DNA. In uninfected Escherichia coli, a ligase is present which utilizes DPN as a cofactor,'-while in phage T4-or T7-infected E. coli an ATPdependent enzyme is induced.7-9 The cofactors are split in both reactions, resulting in the formation of an intermediary enzyme-adenylate complex.3' 8 We have been studying various DNA-modifying enzymes in myeloid and lymphoid tissues, and in the present communication wish to describe the isolation of a polynucleotide ligase from rabbit bone marrow. The enzyme requires ATP as a cofactor and apparently acts by the same general mechanism as microbial ligases.Experimental.-Bone marrow from femoral and tibial bones of adult rabbits (1-3 yr) was obtained from Pel-Freez Biologicals, Inc., Rogers, Arkansas. Other rabbit organs and small amounts of bone marrow were obtained and processed directly after terminal bleeding of the animals by heart puncture.Polynucleotide kinase was purified according to Richardson10 from E. coli B infected with the ligase-defective amber mutant T4 H39X."1 3'-Deoxynucleotidase was purified from the same source.'2 T4-induced polynucleotide ligase was purified from E. coli B infected with T4 am 82.7The following enzymes were commercially obtained: E. coli alkaline phosphatase, pancreatic DNase, micrococcal nuclease, venom phosphodiesterase, spleen phosphodiesterase, yeast alcohol dehydrogenase, catalase, inorganic pyrophosphatase (Worthington), venom 5'-nucleotidase (Sigma), muscle glyceraldehyde 3-phosphate dehydrogenase, and yeast phosphoglycerate kinase (D. F. Boehringer and Soehne). Crystalline bovine serum albumin was purchased from Armour Pharmaceutical Co.Calf thymus DNA was obtained from Worthington. P12-labeled DNA was isolated from E. coli B grown in a Pi32-containing medium;'3 the labeled DNA had an s20. t of 25S in alkaline sucrose, corresponding to an average chain length of 15,000 nucleotides.'4 yP32_ ATP (2 mc/gmole) was prepared according to Glynn and Chappell." The sources of the following compounds were: ATP and DPN, Sigma; dATP, Schwarz BioResearch Inc.; GTP and aged alumina C'y gel, Calbiochem.; 3-phosphoglycerate, C. F. Boehringer and Soehne; phosphocellulose, Whatman; hydroxyapatite, Clarkson Chemical Co.; dextran 500, blue dextran, and Sephadex G-200, Pharmacia Ltd.; polyethylene glycol, type Carbowax 6000, Ruger Chemical Co.; Pi32 and PPi32, New England Nuclear Co. Protein concentrations were determined according to Lowry et al.,6 or in more purified enzyme fractions byabsorbance measurements at 280 mo. DNA concentrations were determined by spectrophotometry, and radioac...

DNA ligases of eukaryotes

“…Reducing agents such as fl-mercaptoethanol or dithiothreitol are necessary for optimal activity, and treatment with parahydroxymercuribenzoate or iV-ethylmaleimide in the absence of such agents causes essentially complete inhibition. The enzyme functions effectively at quite low ATP concentrations, and has an apparent KM of 2 X lo-'-l.5 X low6 [23,24,29,30]. Nevertheless, there is an absolute requirement for ATP, and NAD' or GTP can not serve as alternate cofactors.…”

“…DNA ligase I becomes available. The first report on a DNA ligase in mammalian cells [23] dealt with the properties of the low mol. wt.…”

“…The dominating DNA ligase activity in such organs is DNA ligase I, and consequently most studies on a mammalian DNA ligase have dealt with the properties of this enzyme. It has been partly purified and characterized from rabbit [23], calf [14,15,24-261, human [27,28], mouse [29], rat [ 16,191 and hamster [ 17,301 cells and tissues, but a homogenous enzyme is not yet available. A rabbit antiserum against 1 OOO-fold purified calf thymus DNA ligase I has been shown to equally effectively inhibit the enzyme from other calf tissues as well as the corresponding enzyme from human, mouse, rat and rabbit cells [ 15,191.…”

“…Thus, the mammalian enzyme catalyzes an ATP-dependent pyrophosphate exchange reaction [23], and a covalent enzymeadenylate complex has been isolated after incubation of purified DNA ligase I with radioactive ATP [25]. This ligase-adenylate complex can join single strand breaks in DNA in the absence of ATP, and one adenylate residue is released in each repair event.…”

Deoxyribonuclease Iv: A New Exonuclease From Mammalian Tissues

“…Poly rA-3H (16.7 iuc/.tmole) and poly rU were obtained from Miles Laboratories, Inc. Calf thymus DNA was obtained from Worthington, and 32P-labeled DNA was isolated from E. coli. 16 Aliquots from both samples were reduced to an approximate length of 600 nucleotides per strand by sonic irradiation in a Raytheon well-type sonicator. DNA-3H from human embryonic kidney cells and DNA-'4C from adenovirus type 2 were gifts from Dr. W. Doerfler.…”