Enhanced mitochondrial G-quadruplex formation impedes replication fork progression leading to mtDNA loss in human cells

Abstract

Mitochondrial DNA (mtDNA) replication stalling is considered an initial step in the formation of mtDNA deletions that associate with genetic inherited disorders and aging. However, the molecular details of how stalled replication forks lead to mtDNA deletions accumulation are still unclear. Mitochondrial DNA deletion breakpoints preferentially occur at sequence motifs predicted to form G-quadruplexes (G4s), four-stranded nucleic acid structures that can fold in guanine-rich regions. Whether mtDNA G4s form in vivo and their potential implication for mtDNA instability is still under debate. In here, we developed new tools to map G4s in the mtDNA of living cells. We engineered a G4-binding protein targeted to the mitochondrial matrix of a human cell line and established the mtG4-ChIP method, enabling the determination of mtDNA G4s under different cellular conditions. Our results are indicative of transient mtDNA G4 formation in human cells. We demonstrate that mtDNA-specific replication stalling increases formation of G4s, particularly in the major arc. Moreover, elevated levels of G4 block the progression of the mtDNA replication fork and cause mtDNA loss. We conclude that stalling of the mtDNA replisome enhances mtDNA G4 occurrence, and that G4s not resolved in a timely manner can have a negative impact on mtDNA integrity.

A G-quadruplex-binding platinum complex induces cancer mitochondrial dysfunction through dual-targeting mitochondrial and nuclear G4 enriched genome

“…Given the evidence that mitochondria can be the targets of G4-interactive compounds [8,9] and platinum complexes [37,[48][49][50], we envisioned that Pt-ttpy that combines G4-binding properties and a platinum coordinating moiety, may localize in mitochondria and play a significant role in the mechanisms underlying mitochondrial toxicity. In this line, we raised the questions about whether and how this small molecule affects the processes associated with mitochondrial function based on its unique dual properties.…”

“…G4 structures have been extensively studied in nuclear DNA, mainly clustered in key regions of the genome: telomeres, gene promoters and DNA replication start points [6,7]. Recent emerging evidence emphasized the G4 presence in mitochondrial DNA (mt DNA) as well [8][9][10]. Prediction by the G4 Hunter algorithm revealed that the complete genome of a mitochondrion (16.6 kb) has approximately 96 G4s [4].…”

Absence of both MGME1 and POLG EXO abolishes mtDNA whereas absence of either creates unique mtDNA duplications

“…The CSB2-3 regions contain O H , a GC-rich region which has been described to form G-quadruplex structures ( 1 ). These regions have been reported to have a role in replication stalling ( 28 , 29 , 30 ) and in DNA recombination ( 31 ). In conjunction with the absence of major replicative exonucleases, these G-quadruplexes could be involved in high levels of duplication formation observed in the mutants.…”

G-quadruplex propensity in H. neanderthalensis, H. sapiens and Denisovans mitochondrial genomes

“…Direct evidence for the transient presence of G-quadruplexes in mtDNA was recently obtained by Doimo et al. , using an engineered G4-binding protein targeted to the mitochondrial matrix of a human cell ( 42 ). A comprehensive analyses of mtDNA of various organisms showed that PQS are over-represented in the 3′UTR, D-loops, replication origins and stem loops of mtDNA and G4 sequence in the D-loop is conserved across taxonomic sub-groups ( 43 ).…”