Abstract
DNA ligase I is the major DNA ligase activity in proliferating mammalian cells. The protein has been purified to apparent homogeneity from calf thymus. It has a monomeric structure and a blocked N-terminal residue. DNA ligase I is a 125-kDa polypeptide as estimated by sodium dodecyl sulfate-gel electrophoresis and by gel chromatography under denaturing conditions, whereas hydrodynamic measurements indicate that the enzyme is an asymmetric 98-kDa protein. Immunoblotting with rabbit polyclonal antibodies to the enzyme revealed a single polypeptide of 125 kDa in freshly prepared crude cell extracts of calf thymus. Limited digestion of the purified DNA ligase I with several reagent proteolytic enzymes generated a relatively protease-resistant 85-kDa fragment. This domain retained full catalytic activity. Similar results were obtained with partially purified human DNA ligase I. The active large fragment represents the C-terminal part of the intact protein, and contains an epitope conserved between mammalian DNA ligase I and yeast and vaccinia virus DNA ligases. The function of the N-terminal region of DNA ligase I is unknown.
Highlights
DNA ligase I is the major DNA ligase activity in proliferating mammalian cells
The 50-kDa material did not adsorb to Mono-Q, whereas the 125-kDa protein, the DNA ligase activity, and the enzymeadenylate forming activity coeluted at 0.26 M NaCl during gradient chromatography (Fig. 1)
Analysis of the latter material (Fraction VII) by SDS-polyacrylamide gel electrophoresis (Fig. 2) indicated that DNA ligase I has a molecular mass of 125 kDa and that the preparation was greater than 95% homogeneous
Summary
Cells and Tissues-Calf thymus glands were obtained from newly slaughtered calves (less than 6 months old) at the local abattoir. 500-fold purified from this crude cell extract by ammonium sulfate fractionation, gel filtration, hydroxylapatite chromatography, and DNA-cellulose chromatography as described for the calf thvmus enzyme, with a final yield of 0.2 mg of protein. One unit of DNA ligase activity catalyzes the conversion of 1 nmol of terminal phosphate residues to a phosphatase resistant form in 15 min at 16 “C. contained 60 mM Tris-HCl, pH 8.0, 10 mM MgC12, 5 mM DTT, 50 pg/ml bovine serum albumin, 0.5 pCi of [a-32P]ATP (3000 Ci/mmol, Amersham Corp.), and DNA ligase I. Gel Filtration-Following adenylation, DNA ligase I protein (Fraction VII) was dialyzed at room temperature against 5 M guanidine hydrochloride, 50 mM Tris-HCl, pH 7.5, 10. VII) was carried out at 4 “C on an FPLC Sunerose 12 column (Pharmacia), equilibrated with 50 mM Tris-HCl, pH 7.5,l mM EDTA, 0.5 mM DTT plus 0.2 or 1.0 M NaCl. Fractions containing DNA ligase. The proportion of high molecular mass polypeptide (-125 kDa) detected by silver staining or immunoblotting was quantitated by scanning densitometry
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