mtDNA Recombination: O'Farrell Lab

Mitochondria are the powerhouses of the cell. These organelles support cellular functions through ATP production and have been implicated in diseases ranging from cancer to Parkinson’s. Additionally, their function extends beyond energy. Mitochondria help to control calcium signaling in the cell, and are integral to programed cell death.

Hypothesized to have originated from engulfed bacteria, mitochondria house their own circular DNA, and their health and function relies upon maintaining healthy mitochondrial DNA (mtDNA). In fact, multiple rare genetic diseases arise from mutations in mtDNA, such as Leigh’s syndrome and Leber’s hereditary optic neuropathy.

Recent research has explored genetic treatments for these mtDNA diseases. Mitochondria are maternally inherited – thus “three-parent” embryos were created, combining DNA from two parents, and cytoplasm containing mitochondria from a third. This technique is not yet approved in humans in the US, though it has been approved in the UK to prevent serious mtDNA genetic diseases in embryos. Other work has investigated directly correcting maternal mitochondrial mutations via genome editing techniques.

However, much is still unknown about mtDNA, including an understanding of mitochondrial genetic diversity. In particular, it is unknown whether animal mtDNA undergoes homologous recombination.

Homologous recombination refers to an exchange of DNA sequence between two identical or similar sequences and is an important process for two main reasons. In evolution, it allows for DNA recombination to occur in sperm and eggs creating new sets of genetic traits, contributing to genetic diversity. Secondly, homologous recombination is a key process in repairing breaks in DNA, known as double-strand breaks.

Recent work from the O’Farrell lab at UCSF has discovered evidence for recombination events in mtDNA that reflect both of these purposes.

In a recent publication in ELife on August 3rd, lead author Hansong Ma, Ph.D. of the O’Farrell lab showed that mitochondria in fruit flies are capable of homologous recombination.

To demonstrate this, Ma and O’Farrell took advantage of genetic mutants in combination with the ability to mix recipient and donor mitochondria in early embryos, creating flies carrying diverse mitochondrial genomes each with different genetic mutations. Using this approach, they combined two mutant mitochondria – one that had a temperature sensitive mutation and the other that had impaired transmission.

By placing these mutants at 29˚C, a high enough temperature to trigger the temperature sensitive mutation, and breeding them for several generations, they found some lines that eventually underwent recombination events to create healthy mitochondria. These mitochondria were approximately a 60/40 mix of the two original mtDNAs and lacked both the transmission defects and temperature-sensitive mutations.

Next, Ma and O’Farrell asked whether recombination is stimulated by double strand breaks, as occurs in nuclear DNA. To test this, the authors combined selective pressure with expression of specific restriction enzymes, an enzyme that snips specific parts of DNA, to create cuts in DNA. They found that a higher incidence of double-strand breaks increased the amount of recombination.

Ma’s work indicates that homologous recombination serves a similar purpose in mtDNA as in nuclear DNA. It allows for gene recombination, preserving favorable gene combinations, and repairs double-strand breaks caused by the restriction enzymes.

However, not all homologous recombination serves a beneficial purpose. “If the sequence is repetitive, recombination might happen [in the middle of a gene], increasing the chances of getting a deletion or insertion,” said Ma. Deletions and insertions can be particularly detrimental to gene function. “We know that deletions likely accumulate in mtDNA over the lifespan and during aging, so [recombination] may contribute to this accumulation.”

In addition to a better understanding of mtDNA, this work provides new tools to alter this DNA, a notoriously difficult target for gene manipulation. “When we linearized two mitochondrial genomes [with restriction enzymes], the efficiency of homologous recombination was very high. This led us to think that we could easily develop this as a tool to manipulate the genome,” Ma said.

Creating recombined genomes could lead to new discoveries about mitochondrial genes. “When we introduced a mitochondrial genome from another species [into our flies], it increased the lifespan of the fly by 30%,” said Ma. “If we make a genome with different portions of mtDNA from the other species, then we can map which region is responsible for the enhanced aging.”

This work opens the door for a better understanding of mtDNA, both providing new tools and further elucidating how mtDNA is rearranged and repaired.