The identification and purification of a novel mammalian DNA ligase.
Using a combination of biochemical fractionation and adenylation assays, we have purified a novel 44 kDa protein from human cells which rejoins DNA double-strand breaks. Its rejoining properties and its ability to form an adenylation product with ATP, which can be rapidly dissociated by the presence of DNA breaks, show that this protein is a DNA ligase. As four mammalian DNA ligases have been previously identified we have named this DNA ligase V. Silver staining of the most purified fraction on denaturing polyacrylamide gels reveals a protein doublet of 46/44 kDa of which only the lower band becomes adenylated. Assay of this protein, along with two defined DNA ligases, against DNA templates containing either double and single-strand breaks shows that unlike other DNA ligases, DNA ligase V does not join nicked templates with high efficiency. However, this DNA ligase can join double-strand breaks with a similar efficiency to DNA ligase 1. This result indicates that there may be different types of DNA ligases in mammalian cells which may have specific cellular functions.
Johnson
A P
AP
DNA Repair and Mutagenesis Group, MRC Radiation and Genome Stability Unit, Harwell, Oxon, UK.
Fairman
M P
MP
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology; D002845:Chromatography; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004249:DNA Damage; D011088:DNA Ligases / Q000302:isolation & purification* / Q000378:metabolism*; D004260:DNA Repair; D017886:Durapatite; D006801:Humans; D007668:Kidney; D008322:Mammals; D008970:Molecular Weight; D013379:Substrate Specificity
D017886:Durapatite; D011088:DNA Ligases