D Loops Maintain Mitochondrial Origins

KEY CONCEPTS
- Mitochondria use different origin sequences to initiate replication of each DNA strand.
- Replication of the H strand is initiated in a D loop.
- Replication of the L strand is initiated when its origin is exposed by the movement of the first replication fork.
The origins of replicons in both prokaryotic and eukaryotic chromosomes are static structures: They comprise sequences of DNA that are recognized in duplex form and used to initiate replication at the appropriate time. Initiation requires separating the DNA strands and commencing bidirectional DNA synthesis. A different type of arrangement is found in mitochondria.
Replication begins at a specific origin in the circular duplex DNA. Initially, though, only one of the two parental strands (the H strand in mammalian mitochondrial DNA) is used as a template for synthesis of a new strand. Synthesis proceeds for only a short distance, displacing the original partner (L) strand, which remains single-stranded, as illustrated in FIGURE 1. The condition of this region gives rise to its name as the displacement loop, or D loop.

FIGURE 12.22 The D loop maintains an opening in mammalian mitochondrial DNA, which has separate origins for the replication of each strand.
DNA polymerases cannot initiate synthesis, but require a priming 3′ end (see the chapter DNA Replication). Replication at the H-strand origin is initiated when RNA polymerase transcribes a primer. The 3′ ends are generated in the primer by an endonuclease that cleaves the DNA–RNA hybrid at several discrete sites. The endonuclease is specific for the triple structure of DNA–RNA hybrid plus the displaced DNA single strand. The 3′ end is then extended into DNA by the DNA polymerase.
A single D loop is found as an opening of 500 to 600 bases in mammalian mitochondria. The short strand that maintains the D loop is unstable and turns over; it is frequently degraded and resynthesized to maintain the opening of the duplex at this site.
Some mitochondrial DNAs possess several D loops, reflecting the use of multiple origins. The same mechanism is employed in chloroplast DNA, where (in complex plants) there are two D loops. To replicate mammalian mitochondrial DNA, the short strand in the D loop is extended. The displaced region of the original L strand becomes longer, expanding the D loop. This expansion continues until it reaches a point about two-thirds of the way around the circle. Replication of this region exposes an origin in the displaced L strand. Synthesis of an H strand initiates at this site, which is used by a special primase that synthesizes a short RNA. The RNA is then extended by DNA polymerase, proceeding around the displaced single-stranded L template in the opposite direction from L-strand synthesis.
As a result of the lag in its start, H-strand synthesis has proceeded only a third of the way around the circle when L-strand synthesis finishes. This releases one completed duplex circle and one gapped circle, the latter of which remains partially single-stranded until synthesis of the H strand is completed. Finally, the new strands are sealed to become covalently intact.
The existence of D loops exposes a general principle: An origin can be a sequence of DNA that serves to initiate DNA synthesis using one strand as a template. The opening of the duplex does
not necessarily lead to the initiation of replication on the other strand. In the case of mitochondrial DNA replication, the origins for replicating the complementary strands lie at different locations. Origins that sponsor replication of only one strand are also found in the rolling circle mode of replication .