Plant Organelle Genome Replication

Abstract

Mitochondria and chloroplasts perform essential functions in respiration, ATP production, and photosynthesis, and both organelles contain genomes that encode only some of the proteins that are required for these functions. The proteins and mechanisms for organelle DNA replication are very similar to bacterial or phage systems. The minimal replisome may consist of DNA polymerase, a primase/helicase, and a single-stranded DNA binding protein (SSB), similar to that found in bacteriophage T7. In Arabidopsis, there are two genes for organellar DNA polymerases and multiple potential genes for SSB, but there is only one known primase/helicase protein to date. Genome copy number varies widely between type and age of plant tissues. Replication mechanisms are only poorly understood at present, and may involve multiple processes, including recombination-dependent replication (RDR) in plant mitochondria and perhaps also in chloroplasts. There are still important questions remaining as to how the genomes are maintained in new organelles, and how genome copy number is determined. This review summarizes our current understanding of these processes.

Keywords: DNA repair; DNA replication; chloroplast DNA; plant mitochondrial DNA; recombination-dependent replication (RDR).

Figure 1

Nuclear and organelle genome sizes among different organisms. Mitochondrial genomes (dark bars in upper panel) among animals are compact and remarkably similar in size: ~16.5 kb. Plants, however, have mitochondrial genomes that dwarf those found in animals and vary in size from species to species. Chloroplast genomes vary less in size (open bars in upper panel) from organism to organism but still are relatively large compared to animal mitochondrial genomes. Organelle genome sizes do not correlate with nuclear genome size (bottom panel).

Figure 2

Proposed DNA replication mechanisms for mitochondrial DNA. (A) Rolling circle replication involves unidirectional replication after nicking of one DNA strand. DNA replication continues along the circular molecule displacing the nicked strand. Upon reaching the initial start site, the displaced strand may be nicked and ligated to form a new single stranded circular molecule or synthesis may continue, creating a linear concatemeric molecule which is later converted into multiple single stranded circular copies of the parent molecule. (B) Displacement loop (D-loop) replication proceeds unidirectionally by synthesis of an RNA primer that displaces one of the DNA strands. Upon synthesizing a certain portion of the genome (commonly 2/3) a second origin site is exposed as a single strand, which triggers DNA synthesis in the opposite direction. By the time the first double stranded DNA molecule is finished, synthesis on the parent strand is still ongoing. Once replication reaches the initial start site, the parent strand is displaced as a single stranded circular DNA molecule. The single stranded circular molecules formed by rolling circle and displacement loop replication are later turned into double stranded copies by DNA replication machinery. (C) Recombination-dependent replication (RDR) involves the use of many linear and circular pieces of DNA that share homology. These pieces recombine to form branched linear and “rosette” like intermediates that are copied and replicated by DNA machinery. (D) Electron micrograph image of DNA forming a “rosette” that is likely the result of recombination. (E) Theta replication is so named because of the intermediate it forms as a result of bi-directional DNA replication. Replication initiates bi-directionally at an origin of replication, forming two replication forks. When these replication forks meet, the two double stranded circular molecules are separated. (F) The RITOLS (Ribonucleotide Incorporation ThroughOut the Lagging Strand)/bootlace strategy of replication involves the lagging strand of a replication fork. While the leading strand replicates normally, free pre-synthesized RNA molecules in the mitochondria (indicated by the arrow) hybridize to the lagging strand of the mtDNA starting from the 3’ end of the RNA and proceeding in the 5’ direction. Gaps are filled in and the primers are removed by the DNA replication machinery.

Figure 3

Theoretical model of the plant organellar DNA replisome. Four proteins are most likely involved in the minimal plant organellar DNA replisome, including Pol1A or Pol1B DNA polymerase, Twinkle DNA helicase/primase, and SSB1 single stranded binding protein. This model is similar to the replisome used by T7 phage which includes the proteins gp5 (DNA polymerase), gp4 (helicase/primase), and gp2.5 (single stranded binding protein). Adapted from Wikipedia file: phage T7 replication machinery.png, created 1 February 2015; https://creativecommons.org/licenses/by-sa/4.0/.