Cell cycle-dependent localization and properties of a second mitochondrial DNA ligase in Crithidia fasciculata

The mitochondrial DNA in kinetoplastid protozoa is contained in a single highly condensed structure consisting of thousands of minicircles and approximately 25 maxicircles. The disk-shaped structure is termed kinetoplast DNA (kDNA) and is located in the mitochondrial matrix near the basal body. We have previously identified a mitochondrial DNA ligase (LIG k beta) in the trypanosomatid Crithidia fasciculata that localizes to antipodal sites flanking the kDNA disk where several other replication proteins are localized. We describe here a second mitochondrial DNA ligase (LIG k alpha). LIG k alpha localizes to the kinetoplast primarily in cells that have completed mitosis and contain either a dividing kinetoplast or two newly divided kinetoplasts. Essentially all dividing or newly divided kinetoplasts show localization of LIG k alpha. The ligase is present on both faces of the kDNA disk and at a high level in the kinetoflagellar zone of the mitochondrial matrix. Cells containing a single nucleus show localization of the LIG k alpha to the kDNA but at a much lower frequency. The mRNA level of LIG k alpha varies during the cell cycle out of phase with that of LIG k beta. LIG k alpha transcript levels are maximal during the phase when cells contain two nuclei, whereas LIG k alpha transcript levels are maximal during S phase. The LIG k alpha protein decays with a half-life of 100 min in the absence of protein synthesis. The periodic expression of the LIG k alpha transcript and the instability of the LIG k alpha protein suggest a possible role of the ligase in regulating minicircle replication.