IN-VIVO DETERMINATION OF REPLICATION ORIGINS OF HUMAN MITOCHONDRIAL-DNA BY LIGATION-MEDIATED POLYMERASE CHAIN-REACTION

A large part of replication is aborted in human mitochondria, the resu lt being a D-loop. As few attempts have been made to distinguish free 5' ends of true replicate from those of abortive ones, we examined the 5' ends of true replicate of human mitochondrial DNA at one nucleotid e resolution in vivo by making use of ligation-mediated polymerase cha in reaction. The distribution and relative amounts of origins of the t rue replicate are exactly the same as those of total newly synthesized heavy strands, which means that the abortion of replication is indepe ndent of 5' ends. Treatment of DNA with RNase H frees 5' ends on both heavy and light strands. This is the first in vivo evidence for covale ntly attached primer RNA to nascent strand in human mitochondrial DNA.