Fingerprinting of near-homogeneous DNA ligase I and II from human cells. Similarity of their AMP-binding domains.
DNA ligases play obligatory roles during replication, repair, and recombination. Multiple forms of DNA ligase have been reported in mammalian cells including DNA ligase I, the high molecular mass species which functions during replication, and DNA ligase II, the low molecular mass species which is associated with repair. In addition, alterations in DNA ligase activities have been reported in acute lymphocytic leukemia cells, Bloom's syndrome cells, and cells undergoing differentiation and development. To better distinguish the biochemical and molecular properties of the various DNA ligases from human cells, we have developed a method of purifying multiple species of DNA ligase from HeLa cells by chromatography through DEAE-Bio-Gel, CM-Bio-Gel, hydroxylapatite, Sephacryl S-300, Mono P, and DNA-cellulose. DNA-cellulose chromatography of the partially purified enzymes resolved multiple species of DNA ligase after labeling the enzyme with [alpha-32P]ATP to form the ligase-[32P]AMP adduct. The early eluting enzyme activity (0.25 M NaCl) contained a major 67-kDa-labeled protein, while the late eluting activity (0.48 M NaCl) contained two major labeled proteins of 90 and 78 kDa. Neutralization experiments with antiligase I antibodies indicated that the early and late eluting activity peaks were DNA ligase II and I, respectively. The three major ligase-[32P]AMP polypeptides (90, 78, and 67 kDa) were subsequently purified to near homogeneity by elution from preparative sodium dodecyl sulfate-polyacrylamide gels. All three polypeptides retained DNA ligase activities after gel elution and renaturation. To further reveal the relationship between these enzymes, partial digestion by V8-protease was performed. All three purified polypeptides gave rise to a common 22-kDa-labeled fragment for their AMP-binding domains, indicating that the catalytic sites of ligase I and II are quite similar, if not identical. Similar findings were obtained from the two-dimensional gel electrophoresis of their AMP-binding domains in the trypsin-digested protein fragments. The results also suggested that these isozymes have been derived from the same primordial DNA sequence or from the same precursor protein. The purification scheme and the data obtained will be instrumental for the further elucidation of the biological roles of various DNA ligases from human cells.
Department of Molecular Pathology, University of Texas M. D. Anderson Cancer Center, Houston 77030.
S W
SW
Yang
F F
FF
Becker
J Y
JY
Chan
D000249:Adenosine Monophosphate / Q000378:metabolism; D001665:Binding Sites; D011088:DNA Ligases / Q000737:chemistry / Q000302:isolation & purification*; D015180:Electrophoresis, Gel, Two-Dimensional; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D007526:Isoelectric Point; D008970:Molecular Weight; D010449:Peptide Mapping; D012697:Serine Endopeptidases / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
D000249:Adenosine Monophosphate; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin; D011088:DNA Ligases