Longley is an associate scientist in the Mitochondrial DNA replication group, led by Bill Copeland, Ph.D. The group studies how replication produces and prevents mtDNA mutations. (Photo courtesy of Steve McCaw)

NIEHS research refining the model of mitochondrial DNA (mtDNA) replication suggests a mechanism that leads to deletions in mtDNA, according to Matt Longley, Ph.D., from the NIEHS Mitochondrial DNA Replication Group. Advances in this field may provide clues to diseases that affect tissues throughout the body, which result from defects in mtDNA.

Longley spoke at Duke University Jan. 25 as part of the University Program in Environmental Health and Toxicology Seminar Series. His talk was titled 'Single-Molecule Imaging of Mitochondrial DNA Replication Proteins Suggest a Mechanism for mtDNA Deletions.'

Mitochondria — essential and uniquely susceptible

Mitochondria, which are the parts of cells responsible for energy production, are especially vulnerable to mutations, Longley explained. Unlike other organelles, mitochondria have their own specialized DNA.

Longley was a postdoctoral fellow in the lab of Paul Modrich, Ph.D., James B. Duke Professor of Biochemistry at Duke University. Modrich was a winner of the 2015 Nobel Prize in Chemistry for his work on DNA mismatch repair. (Photo courtesy of Steve McCaw)

Numerous environmental chemicals, such as polycyclic aromatic hydrocarbons and metals, can damage mtDNA and lead to mutations. “Unlike the nucleus, mitochondria cannot execute nucleotide excision repair,” Longley said.

The lack of certain DNA repair mechanisms means that mitochondria cannot always fix the damage caused by exposures. Moreover, mtDNA does not undergo genetic recombination, which helps cells to weed out harmful mutations in nuclear DNA.

Refining the mtDNA replication model

Longley and colleagues focused on two proteins in the human mtDNA replication process — mitochondrial single-strand DNA binding (mtSSB) protein and Twinkle helicase.

Replication requires mtDNA to unwind and unzip into single-stranded DNA. During this vulnerable stage, mtSSB protects the exposed mtDNA strand from damage that can promote mutations. Imaging such molecular events is no easy task, so Longley and his colleagues collaborated with researchers at North Carolina State University (NCSU) to use innovative microscopy techniques.

Students and others packed Field Auditorium to hear Longley’s presentation. (Photo courtesy of Steve McCaw)

Single-molecule imaging and atomic force microscopy (AFM) enabled the researchers to look at mtDNA replication proteins at resolutions of only a few nanometers. Using a technology called Dual Resonance Frequency Enhanced Electrostatic Microscopy (DREEM), they took pictures of mtDNA wrapping around mtSSB.

Techniques like DREEM and AFM enable better imaging of DNA within protein-DNA complexes, helping scientists build understanding of DNA replication. According to Longley, the conclusions from this research, not yet published, indicate that mtSSB may not fully protect single-stranded mtDNA during replication. This may be a key factor in forming mtDNA deletions that give rise to mitochondrial diseases.

(Former NIEHS postbaccalaureate fellow Samantha Hall is a graduate student at Duke University and an NIEHS Special Volunteer.)