Mitochondrial DNA replication in human T lymphocytes is regulated primarily at the H-strand termination site.
The most unique feature in the replication of mitochondrial DNA (mtDNA) is that most of the newly synthesized heavy strands (H-strands) terminate prematurely, resulting in the formation of displacement loop (D-loop) strands. Only the H-strand which proceeds past the termination site is a true nascent H-strand leading to the overall replication on a circular mtDNA molecule. The physiological significance of the D-loop formation has long been unclear. To examine the role of premature termination in mtDNA replication, we therefore developed a method for selectively measuring both the total amount of nascent H-strands and the amount of true nascent H-strands using ligation-mediated polymerase chain reaction, which, for the first time, enabled us to estimate the frequency of premature termination. The stimulation of cell proliferation with interleukin 2 and phytohemagglutinin in human peripheral T lymphocytes caused an increase in the net replication rate of mtDNA. In stimulated cells, in comparison to resting ones, the amount of true nascent H-strands increased approx. 2.6-fold while the total amount of nascent H-strands remained unchanged, indicating that premature termination decreased while the initiation of replication remained the same. Our findings thus demonstrate the first clear example that premature termination plays a primary role in the up-regulation of the net rate of mtDNA replication in human cells.
Kai
Y
Y
Department of Biochemistry, Kyushu University School of Medicine, Fukuoka 812-8582, Japan.
Miyako
K
K
Muta
T
T
Umeda
S
S
Irie
T
T
Hamasaki
N
N
Takeshige
K
K
Kang
D
D
D016590:Aphidicolin / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004261:DNA Replication / Q000235:genetics*; D004272:DNA, Mitochondrial / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D019169:Jurkat Cells; D008934:Mitogens / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D015854:Up-Regulation
D004272:DNA, Mitochondrial; D004791:Enzyme Inhibitors; D008934:Mitogens; D016590:Aphidicolin