6. D loops maintain mitochondrial origins

12.6 D loops maintain mitochondrial origins

Key terms defined in this section

D loop is a region within mitochondrial DNA in which a short stretch of RNA is paired with one strand of DNA, displacing the original partner DNA strand in this region. The same term is used also to describe the displacement of a region of one strand of duplex DNA by a single-stranded invader in the reaction catalyzed by RecA protein.

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.

Figure 12.11 The D loop maintains an opening in mammalian mitochondrial DNA, which has separate origins for the replication of each strand.

Replication starts at a specific origin in the circular duplex DNA. But initially 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 12.11. The condition of this region gives rise to its name as the displacement or D loop (for review see Clayton, 1982; Clayton, 1991).

DNA polymerases cannot initiate synthesis, but require a priming 3′ end (see 13 DNA replication). Replication at the H strand origin is initiated in the usual way, by synthesis of an RNA primer. RNA polymerase transcribes a primer, whose 3′ ends are generated by cleavage by an endonuclease 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 V600 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 higher 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.

Because 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, which remains partially single-stranded until synthesis of the H strand is completed. Finally, the new strands are sealed to become covalently intact (for review see Shadel and Clayton, 1997).

The existence of rolling circles and D loops exposes a general principle. An origin can be a sequence of DNA that serves to initiate DNA synthesis using one strand as 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.

Reviews

Clayton, D., A. (1991). Replication and transcription of vertebrate mitochondrial DNA. Ann. Rev. Cell Biol. 7, 453-478.

Clayton, D. (1982). Replication of animal mitochondrial DNA. Cell 28, 693-705.

Shadel, G. S. and Clayton, D. A. (1997). Mitochondrial DNA maintenance in vertebrates. Ann. Rev. Biochem 66, 409-435.