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 prem aturely, resulting in the formation of displacement loop (D-loop) strands. Only the H-strand which proceeds past the termination site is a true nascen t H-strand leading to the overall replication on a circular mtDNA molecule. The physiological significance of the D-loop formation has long been uncle ar. To examine the role of premature termination in mtDNA replication, we t herefore developed a method for selectively measuring both the total amount of nascent H-strands and the amount of true nascent H-strands using Ligati on-mediated polymerase chain reaction, which, for the first time, enabled u s to estimate the frequency of premature termination. The stimulation of ce ll proliferation with interleukin 2 and phytohemagglutinin in human periphe ral T lymphocytes caused an increase in the net replication rate of mtDNA. In stimulated cells, in comparison to resting ones, the amount of true nasc ent H-strands increased approx. 2.6-fold while the total amount of nascent H-strands remained unchanged, indicating that premature termination decreas ed while the initiation of replication remained the same. Our findings thus demonstrate the first clear example that premature termination plays a pri mary role in the up-regulation of the net rate of mtDNA replication in huma n cells. (C) 1999 Elsevier Science B.V. All rights reserved.