Biology subjects

Kavlashvili, T.

Publications and source records attributed to Kavlashvili, T..

7 recordsLinked to original sources

bioRxiv2026

Respiration-Deficient Cells Require Pyruvate Carboxylase to Suppress Asparagine Auxotrophy

Mutations in mitochondrial DNA (mtDNA) compromise ETC activity and impair oxidative phosphorylation. Since eukaryotic cells contain multiple copies of mtDNA, the resulting phenotype depends on the proportion of mutant mitochondrial genomes (the heteroplasmy level). Using isogenic cell lines carrying similar mtDNA deletions, a linear decline in cellular respiration was observed as mitochondrial DNA heteroplasmy increased. Despite this, cellular redox imbalance did not change until heteroplasmy exceeded 50%. As heteroplasmy increased past 70%, cells also exhibited an integrated stress response (ISR) and impaired translation was observed. These defects were reversed by either addition of asparagine or overexpression of pyruvate carboxylase (PC). The dependence on exogenous asparagine in other respiration-deficient cells was found to correlate inversely with the PC expression level. For example, patient-derived thyroid tumor cells, harboring high heteroplasmy for a Complex I mtDNA mutation and low levels of PC, exhibited asparagine auxotrophy, and L-asparaginase treatment suppressed tumor growth. Together, these findings demonstrate a role for mitochondrial pyruvate carboxylase in cellular asparagine synthesis under conditions of compromised respiratory activity.

cell biology↗

bioRxiv2026

MTERF1 loss buffers against pathogenic mtDNA deletions through transcriptional regulation

Large-scale mitochondrial DNA (mtDNA) deletions cripple oxidative phosphorylation once they exceed a critical heteroplasmy threshold, causing incurable mitochondrial pathologies. Using a genome-wide CRISPR/Cas9 screen in an engineered human cell line carrying a large-scale mtDNA deletion at high heteroplasmy, we identified mitochondrial transcription termination factor 1 (MTERF1) as a suppressor of the heteroplasmy burden. Loss of MTERF1 restored mitochondrial function and increased cellular proliferation in cells with a mtDNA deletion burden exceeding the pathogenic threshold, without altering heteroplasmy or mtDNA copy number. MTERF1 binds wild-type and deletion-bearing mitochondrial genomes indiscriminately at a site downstream of the ribosomal RNA genes and curbs transcription. Relieving this constraint broadly increased OXPHOS transcripts, thereby eliciting more respiratory output from the residual wild-type genomes. Notably, the buffering effect of MTERF1 loss extended beyond mtDNA deletions. In a counter-screen, MTERF1 loss could also restore respiratory growth in cells depleted of nuclear-encoded mitochondrial genes such as OPA1 and COX5A. Together, these findings indicate that by relieving a transcriptional constraint, MTERF1 loss compensates for reduced genome dosage, defining a strategy to enhance residual mitochondrial function in mtDNA deletion disorders and related conditions.

molecular biology↗

bioRxiv2026

Strand-independent degradation of uncoupled forks by EXO1 activates ATR and restrains synthesis

Nascent DNA degradation of replication forks shapes genome stability, yet the mechanism of degradation, and its direct consequences, remain unclear. Here we use localized nascent strand degradation at replication forks in Xenopus egg extracts to examine the mechanism and consequences of degradation. The exonuclease EXO1 is crucial for degradation and acts specifically on replication fork structures. Degradation requires EXO1 catalytic activity and is defective in the E109K Lynch Syndrome associated mutant. EXO1 degrades both nascent strands 5-3: the lagging strand from its native 5 end and the leading strand from a distal 5 entry on the sister fork, whereas leading strand 3 end remains stable. Impaired leading-strand degradation at a specific site does not affect degradation of the corresponding lagging-strand region, so the two strands are degraded independently. Degradation of the uncoupled fork has two downstream consequences. It is important to activate ATR, which is otherwise weakly activated by the uncoupled fork. Additionally, degradation restrains fork progression, independent of ATR activation. Our findings demonstrate that strand-independent degradation of uncoupled forks by EXO1 activates ATR and restrains fork progression

biochemistry↗

bioRxiv2024

Engineering mtDNA Deletions by Reconstituting End-Joining in Human Mitochondria

Recent breakthroughs in the genetic manipulation of mitochondrial DNA (mtDNA) have enabled the precise introduction of base substitutions and the effective removal of genomes carrying harmful mutations. However, the reconstitution of mtDNA deletions responsible for severe mitochondrial myopathies and age-related diseases has not yet been achieved in human cells. Here, we developed a method to engineer specific mtDNA deletions in human cells by co-expressing end-joining (EJ) machinery and targeted endonucleases. As a proof-of-concept, we used mito-EJ and mito-ScaI to generate a panel of clonal cell lines harboring a [~]3.5 kb mtDNA deletion with the full spectrum of heteroplasmy. Investigating these isogenic cells revealed a critical threshold of [~]75% deleted genomes, beyond which cells exhibited depletion of OXPHOS proteins, severe metabolic disruption, and impaired growth in galactose-containing media. Single-cell multiomic analysis revealed two distinct patterns of nuclear gene deregulation in response to mtDNA deletion accumulation; one triggered at the deletion threshold and another progressively responding to increasing heteroplasmy. In summary, the co-expression of mito-EJ and programable nucleases provides a powerful tool to model disease-associated mtDNA deletions in different cell types. Establishing a panel of cell lines with a large-scale deletion at varying levels of heteroplasmy is a valuable resource for understanding the impact of mtDNA deletions on diseases and guiding the development of potential therapeutic strategies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=143 HEIGHT=200 SRC="FIGDIR/small/618543v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@ab900corg.highwire.dtl.DTLVardef@17e094dorg.highwire.dtl.DTLVardef@194a10corg.highwire.dtl.DTLVardef@d925e5_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LICombining prokaryotic end-joining with targeted endonucleases generates specific mtDNA deletions in human cells C_LIO_LIEngineering a panel of cell lines with a large-scale deletion that spans the full spectrum of heteroplasmy C_LIO_LI75% heteroplasmy is the threshold that triggers mitochondrial and cellular dysfunction C_LIO_LITwo distinct nuclear transcriptional programs in response to mtDNA deletions: threshold-triggered and heteroplasmy-sensing C_LI

molecular biology↗

bioRxiv2022

RTEL1 and MCM10 overcome topological stress during vertebrate replication termination

Topological stress can cause replication forks to stall as they converge upon one another during termination of vertebrate DNA synthesis. However, replication forks ultimately overcome topological stress and complete DNA synthesis, suggesting that alternative mechanisms can overcome topological stress. We performed a proteomic analysis of converging replication forks that were stalled by topological stress in Xenopus egg extracts. We found that the helicase RTEL1 and the replisome protein MCM10 were highly enriched on DNA under these conditions. We show that RTEL1 normally plays a minor role during fork convergence while the role of MCM10 is normally negligible. However, RTEL1 and MCM10 both become crucially important for fork convergence under conditions of topological stress. RTEL1 and MCM10 exert non-additive effects on fork convergence and physically interact, suggesting that they function together. Furthermore, RTEL1 and MCM10 do not impact topoisomerase activity but do promote fork progression through a replication barrier. Thus, RTEL1 and MCM10 appear to play a general role in promoting progression of stalled forks, including when forks stall during termination. Overall, our data identify an alternate mechanism of termination involving RTEL1 and MCM10 that can be used to complete DNA synthesis under conditions of topological stress.

biochemistry↗

bioRxiv2021

Replication Fork Uncoupling Causes Nascent Strand Degradation and Fork Reversal

Genotoxins cause nascent strand degradation (NSD) and fork reversal during DNA replication. NSD and fork reversal are crucial for genome stability and exploited by chemotherapeutic approaches. However, it is unclear how NSD and fork reversal are triggered. Additionally, the fate of the replicative helicase during these processes is unknown. We developed a biochemical approach to study synchronous, localized NSD and fork reversal using Xenopus egg extracts. We show that replication fork uncoupling stimulates NSD of both nascent strands and progressive conversion of uncoupled forks to reversed forks. The replicative helicase remains bound during NSD and fork reversal, indicating that both processes take place behind the helicase. Unexpectedly, NSD occurs before and after fork reversal, indicating that multiple degradation steps take place. Overall, our data show that uncoupling causes NSD and fork reversal and identify key steps involved in these processes.

biochemistry↗

bioRxiv2021

Topoisomerase II poisons inhibit vertebrate DNA replication through distinct mechanisms

Topoisomerase II (Top2) unlinks chromosomes during vertebrate DNA replication. Top2 poisons are widely-used chemotherapeutics that stabilize Top2 complexes on DNA, leading to cytotoxic DNA breaks. However, it is unclear how these drugs affect DNA replication, which is a major target of Top2 poisons. Using Xenopus egg extracts, we show that the Top2 poisons etoposide and doxorubicin both inhibit DNA replication through different mechanisms. Etoposide induces Top2-dependent DNA breaks and induces Top2-dependent fork stalling by trapping Top2 behind replication forks. In contrast, doxorubicin does not lead to appreciable break formation and instead intercalates into parental DNA to inhibit replication fork progression. In human cells, etoposide stalls replication forks in a Top2-dependent manner, while doxorubicin stalls forks independently of Top2. However, both drugs exhibit Top2-dependent cytotoxicity. Thus, despite shared genetic requirements for cytotoxicity etoposide and doxorubicin inhibit DNA replication through distinct mechanisms.

biochemistry↗