Trypanosomes have six mitochondrial DNA helicases with one controlling kinetoplast maxicircle replication.
Kinetoplast DNA (kDNA), the trypanosome mitochondrial DNA, contains thousands of minicircles and dozens of maxicircles interlocked in a giant network. Remarkably, Trypanosoma brucei's genome encodes 8 PIF1-like helicases, 6 of which are mitochondrial. We now show that TbPIF2 is essential for maxicircle replication. Maxicircle abundance is controlled by TbPIF2 level, as RNAi of this helicase caused maxicircle loss, and its overexpression caused a 3- to 6-fold increase in maxicircle abundance. This regulation of maxicircle level is mediated by the TbHslVU protease. Previous experiments demonstrated that RNAi knockdown of TbHslVU dramatically increased abundance of minicircles and maxicircles, presumably because a positive regulator of their synthesis escaped proteolysis and allowed synthesis to continue. Here, we found that TbPIF2 level increases following RNAi of the protease. Therefore, this helicase is a TbHslVU substrate and an example of a positive regulator, thus providing a molecular mechanism for controlling maxicircle replication.
Liu
Beiyu
B
Department of Biological Chemistry, Johns Hopkins Medical School, Baltimore, MD 21205, USA.
Wang
Jianyang
J
Yaffe
Nurit
N
Lindsay
Megan E
ME
Zhao
Zhixing
Z
Zick
Aviad
A
Shlomai
Joseph
J
Englund
Paul T
PT
D000818:Animals; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D018105:DNA, Kinetoplast / Q000096:biosynthesis*; D004272:DNA, Mitochondrial / Q000096:biosynthesis*; D016054:DNA, Protozoan / Q000096:biosynthesis*; D005786:Gene Expression Regulation; D009154:Mutation; D010447:Peptide Hydrolases / Q000378:metabolism; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D013997:Time Factors; D014162:Transfection; D014346:Trypanosoma brucei brucei / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development
D018105:DNA, Kinetoplast; D004272:DNA, Mitochondrial; D016054:DNA, Protozoan; D015800:Protozoan Proteins; D010447:Peptide Hydrolases; D004265:DNA Helicases