Purification of DNA ligases from mouse testis and their behavior during meiosis.

Abstract

ABSTRACT. Two types of DNAligase, I and II, have been purified approximately 4,000-fold from mouse testes and 500-fold from nuclei of mouse spermatocytes. DNAligase I and II consisted of single polypeptides with molecular weights of 95,000 and 65,000, respectively, according to the estimation by SDS-polyacrylamide gel electrophoresis and the AMP-bindingassay. Ligase activities were higher in premeiotic spermatogonia and spermatocytes than those in liver and bone marrow cells. Moreover, DNAligase II showed rapid increase during meiotic prophase and a decrease in round spermatids. Since this behavior of DNAligase II is consistent with that of m-rec and DNApolymerase fi, both of which have been shown to be involved in DNArecombination in meiotic cells, DNAligase II might be an enzyme which works at the final step of meiotic recombination reaction.

Mammalian DNA Ligases

“…This was the case for the first report on a mammalian DNA ligase activity (24) as well as for several more recent investigations (25)(26)(27)(28)(29). Other early studies reported on both the intact fonn and the active fragment of DNA ligase I (30).…”

“…A DNA ligase distinct from DNA ligase I is more firmly associated with cell nuclei and is only extracted into soluble form by salt-containing buffers (27,39,70). The enzyme has been purified from calf thymus as a 68-72-kDa protein of apparent homogeneity, estimated by SDS-polyacrylamide gel elec trophoresis, by three different groups (27,45,71).…”

“…The enzyme has been purified from calf thymus as a 68-72-kDa protein of apparent homogeneity, estimated by SDS-polyacrylamide gel elec trophoresis, by three different groups (27,45,71). The protein has a blocked N-terminal residue.…”

“…DNA ligase II and ezrin/cytovillin are of the same size, and these two proteins also share the unusual property of adsorbing very tenaciously to hydroxylapatite, which consequently is a key purification step for either protein (45,75,76). Probably the easiest remedy for this copurification problem is to isolate DNA ligase II from cell nuclei rather than from whole cell extracts (27). The presence of variable amounts of ezrinlcytovillin in DNA ligase II preparations is unlikely to affect the catalytic properties of the DNA ligase, however, and the ezrin/cytovillin may even serve as a carrier protein instead of externally added bovine serum albumin.…”

“…Furthermore, the presence of the enzyme in the postmicrosomal supernatant of rat liver cells extracted with an isotonic su crose buffer indicates that the enzyme has a similar subcellular distribution to DNA ligase I, which leaches out of nuclei under such conditions. In contrast, DNA ligase II is not extracted from nuclei with isotonic sucrose (27,39,70).…”