Increases in mitochondrial DNA content and 4977-bp deletion upon ATM/Chk2 checkpoint activation in HeLa cells

Activation of the Mec1/Rad53 damage checkpoint pathway influences mitochondrial DNA (mtDNA) content and point mutagenesis in Saccharomyces cerevisiae. The effects of this conserved checkpoint pathway on mitochondrial genomes in human cells remain largely unknown. Here, we report that knockdown of the human DNA helicase RRM3 enhances phosphorylation of the cell cycle arrest kinase Chk2, indicating activation of the checkpoint via the ATM/Chk2 pathway, and increases mtDNA content independently of TFAM, a regulator of mtDNA copy number. Cell-cycle arrest did not have a consistent effect on mtDNA level: knockdown of cell cycle regulators PLK1 (polo-like kinase), MCM2, or MCM3 gave rise, respectively, to decreased, increased, or almost unchanged mtDNA levels. Therefore, we concluded that the mtDNA content increase upon RRM3 knockdown is not a response to delay of cell cycle progression. Also, we observed that RRM3 knockdown increased the levels of reactive oxygen species (ROS); two ROS scavengers, N-acetyl cysteine and vitamin C, suppressed the mtDNA content increase. On the other hand, in RRM3 knockdown cells, we detected an increase in the frequency of the common 4977-bp mtDNA deletion, a major mtDNA deletion that can be induced by abnormal ROS generation, and is associated with a decline in mitochondrial genome integrity, aging, and various mtDNA-related disorders in humans. These results suggest that increase of the mitochondrial genome by TFAM-independent mtDNA replication is connected, via oxidative stress, with the ATM/Chk2 checkpoint activation in response to DNA damage, and is accompanied by generation of the common 4977-bp deletion.     

A Heterozygous Truncating Mutation in RRM2B Causes Autosomal-Dominant Progressive External Ophthalmoplegia with Multiple mtDNA Deletions

Autosomal-dominant progressive external ophthalmoplegia (adPEO) is a mitochondrial disorder that is characterized by accumulation of multiple mitochondrial DNA (mtDNA) deletions in postmitotic tissues. The disorder is heterogeneous, with five known nuclear disease genes that encode the proteins ANT1, Twinkle, POLG, POLG2, and OPA1. Defects in these proteins affect mtDNA maintenance, probably leading to stalled replication forks, consequent mtDNA deletion formation, and progressive respiratory chain deficiency. Here we present a large adPEO family with multiple mtDNA deletions, whose disease was not explained by mutations in any of the known adPEO loci. We mapped the disease locus in this family to chromosome 8q22.1-q23.3. The critical linkage region contained the RRM2B gene, which encodes the small subunit of the ribonucleotide reductase p53R2, which has previously been shown to be essential for the maintenance of mtDNA copy number. Mutation screening of RRM2B revealed a heterozygous nonsense mutation in exon 9 (c.979C→T [p.R327X]) in all affected individuals that was absent in 380 control chromosomes. The same mutation was found to segregate in another adPEO family. The mutant mRNA escaped nonsense-mediated decay and resulted in a protein with truncation of 25 highly conserved C-terminal amino acids essential for the interaction with the ribonucleotide reductase subunit R1. We conclude that dominant-negative or gain-of-function mutations in RRM2B are a cause of multiple mtDNA deletions and adPEO.     

Mec1p is essential for phosphorylation of the yeast DNA damage checkpoint protein Ddc1p, which physically interacts with Mec3p.

Checkpoints prevent DNA replication or nuclear division when chromosomes are damaged. The Saccharomyces cerevisiae DDC1 gene belongs to the RAD17, MEC3 and RAD24 epistasis group which, together with RAD9, is proposed to act at the beginning of the DNA damage checkpoint pathway. Ddc1p is periodically phosphorylated during unperturbed cell cycle and hyperphosphorylated in response to DNA damage. We demonstrate that Ddc1p interacts physically in vivo with Mec3p, and this interaction requires Rad17p. We also show that phosphorylation of Ddc1p depends on the key checkpoint protein Mec1p and also on Rad24p, Rad17p and Mec3p. This suggests that Mec1p might act together with the Rad24 group of proteins at an early step of the DNA damage checkpoint response. On the other hand, Ddc1p phosphorylation is independent of Rad53p and Rad9p. Moreover, while Ddc1p is required for Rad53p phosphorylation, it does not play any major role in the phosphorylation of the anaphase inhibitor Pds1p, which requires RAD9 and MEC1. We suggest that Rad9p and Ddc1p might function in separated branches of the DNA damage checkpoint pathway, playing different roles in determining Mec1p activity and/or substrate specificity.     

Mdt1, a novel Rad53 FHA1 domain-interacting protein, modulates DNA damage tolerance and G(2)/M cell cycle progression in Saccharomyces cerevisiae

The Rad53 kinase plays a central role in yeast DNA damage checkpoints. Rad53 contains two FHA phosphothreonine-binding domains that are required for Rad53 activation and possibly downstream signaling. Here we show that the N-terminal Rad53 FHA1 domain interacts with the RNA recognition motif, coiled-coil, and SQ/TQ cluster domain-containing protein Mdt1 (YBl051C). The interaction of Rad53 and Mdt1 depends on the structural integrity of the FHA1 phosphothreonine-binding site as well as threonine-305 of Mdt1. Mdt1 is constitutively threonine phosphorylated and hyperphosphorylated in response to DNA damage in vivo. DNA damage-dependent Mdt1 hyperphosphorylation depends on the Mec1 and Tel1 checkpoint kinases, and Mec1 can directly phosphorylate a recombinant Mdt1 SQ/TQ domain fragment. MDT1 overexpression is synthetically lethal with a rad53 deletion, whereas mdt1 deletion partially suppresses the DNA damage hypersensitivity of checkpoint-compromised strains and generally improves DNA damage tolerance. In the absence of DNA damage, mdt1 deletion leads to delayed anaphase completion, with an elongated cell morphology reminiscent of that of G(2)/M cell cycle mutants. mdt1-dependent and DNA damage-dependent cell cycle delays are not additive, suggesting that they act in the same pathway. The data indicate that Mdt1 is involved in normal G(2)/M cell cycle progression and is a novel target of checkpoint-dependent cell cycle arrest pathways.     

Saccharomyces cerevisiae Rad9 acts as a Mec1 adaptor to allow Rad53 activation

BACKGROUND: The DNA damage checkpoint is a protein kinase-based signaling system that detects and signals physical alterations in DNA. Despite having identified many components of this signaling cascade, the exact mechanisms by which checkpoint kinases are activated after DNA damage, as well as the role of the checkpoint mediators, remain poorly understood. RESULTS: To elucidate the mechanisms that underlie the MEC1 and RAD9-dependent activation of Rad53, the Saccharomyces cerevisiae ortholog of Chk2, we mapped and characterized in vivo phosphorylation sites present on Rad53 after DNA damage by mass spectrometry. We find that Rad53 requires for its activation multisite phosphorylation on a number of typical and atypical Mec1 phosphorylation sites, thus confirming that Rad53 is a direct target of Mec1, the mammalian ATR homolog. Moreover, by using biochemical reconstitution experiments, we demonstrate that efficient and direct phosphorylation of Rad53 by Mec1 is only observed in the presence of purified Rad9, the archetypal checkpoint mediator. We find that the stimulatory activity of Rad9 requires a phospho- and FHA-dependent interaction with Rad53, which allows Rad53 to be recognized as a substrate for Mec1. CONCLUSIONS: Our results indicate that Rad9 acts as a bona fide signaling adaptor that enables Rad53 phosphorylation by Mec1. Given the high degree of conservation of checkpoint signaling in eukaryotes, we propose that one of the critical functions of checkpoint mediators such as MDC1, 53BP1, or Brca1 is to act as PIKK adaptors during the DNA damage response.     

Mec1 and Rad53 inhibit formation of single-stranded DNA at telomeres of Saccharomyces cerevisiae cdc13-1 mutants

Here we examine the roles of budding-yeast checkpoint proteins in regulating degradation of dsDNA to ssDNA at unprotected telomeres (in Cdc13 telomere-binding protein defective strains). We find that Rad17, Mec3, as well as Rad24, members of the putative checkpoint clamp loader (Rad24) and sliding clamp (Rad17, Mec3) complexes, are important for promoting degradation of dsDNA in and near telomere repeats. We find that Mec1, Rad53, as well as Rad9, have the opposite role: they inhibit degradation. Downstream checkpoint kinases Chk1 and Dun1 play no detectable role in either promoting degradation or inhibiting it. These data suggest, first, that the checkpoint sliding clamp regulates and/or recruits some nucleases for degradation, and, second, that Mec1 activates Rad9 to activate Rad53 to inhibit degradation. Further analysis shows that Rad9 inhibits ssDNA generation by both Mec1/Rad53-dependent and -independent pathways. Exo1 appears to be targeted by the Mec1/Rad53-dependent pathway. Finally, analysis of double mutants suggests a minor role for Mec1 in promoting Rad24-dependent degradation of dsDNA. Thus, checkpoint proteins orchestrate carefully ssDNA production at unprotected telomeres.     

A Ddc2-Rad53 fusion protein can bypass the requirements for RAD9 and MRC1 in Rad53 activation

Activation of Rad53p by DNA damage plays an essential role in DNA damage checkpoint pathways. Rad53p activation requires coupling of Rad53p to Mec1p through a “mediator” protein, Rad9p or Mrc1p. We sought to determine whether the mediator requirement could be circumvented by making fusion proteins between the Mec1 binding partner Ddc2p and Rad53p. Ddc2-Rad53p interacted with Mec1p and other Ddc2-Rad53p molecules under basal conditions and displayed an increased oligomerization upon DNA damage. Ddc2-Rad53p was activated in a Mec1p- and Tel1p-dependent manner upon DNA damage. Expression of Ddc2-Rad53p in Δrad9 or Δrad9Δmrc1 cells increased viability on plates containing the alkylating agent methyl methane sulfonate. Ddc2-Rad53p was activated at least partially by DNA damage in Δrad9Δmrc1 cells. In addition, expression of Ddc2-Rad53p in Δrad24Δrad17Δmec3 cells increased cell survival. These results reveal minimal requirements for function of a core checkpoint signaling system.     

The checkpoint clamp activates Mec1 kinase during initiation of the DNA damage checkpoint

Yeast Mec1/Ddc2 protein kinase, the ortholog of human ATR/ATRIP, plays a central role in the DNA damage checkpoint. The PCNA-like clamp Rad17/Mec3/Ddc1 (the 9-1-1 complex in human) and its loader Rad24-RFC are also essential components of this signal transduction pathway. Here we have studied the role of the clamp in regulating Mec1, and we delineate how the signal generated by DNA lesions is transduced to the Rad53 effector kinase. The checkpoint clamp greatly activates the kinase activity of Mec1, but only if the clamp is appropriately loaded upon partial duplex DNA. Activated Mec1 phosphorylates the Ddc1 and Mec3 subunits of the clamp, the Rad24 subunit of the loader, and the Rpa1 and Rpa2 subunits of RPA. Phosphorylation of Rad53, and of human PHAS-1, a nonspecific target, also requires a properly loaded clamp. Phosphorylation and binding studies with individual clamp subunits indicate that the Ddc1 subunit mediates the functional interactions with Mec1.     

Role of a Complex Containing Rad17, Mec3, and Ddc1 in the Yeast DNA Damage Checkpoint Pathway

Genetic analysis has suggested that RAD17, RAD24, MEC3, and DDC1 play similar roles in the DNA damage checkpoint control in budding yeast. These genes are required for DNA damage-induced Rad53 phosphorylation and considered to function upstream of RAD53 in the DNA damage checkpoint pathway. Here we identify Mec3 as a protein that associates with Rad17 in a two-hybrid screen and demonstrate that Rad17 and Mec3 interact physically in vivo. The amino terminus of Rad17 is required for its interaction with Mec3, and the protein encoded by the rad17-1 allele, containing a missense mutation at the amino terminus, is defective for its interaction with Mec3 in vivo. Ddc1 interacts physically and cosediments with both Rad17 and Mec3, indicating that these three proteins form a complex. On the other hand, Rad24 is not found to associate with Rad17, Mec3, and Ddc1. DDC1 overexpression can partially suppress the phenotypes of the rad24Δ mutation: sensitivity to DNA damage, defect in the DNA damage checkpoint and decrease in DNA damage-induced phosphorylation of Rad53. Taken together, our results suggest that Rad17, Mec3, and Ddc1 form a complex which functions downstream of Rad24 in the DNA damage checkpoint pathway.     

β-lapachone Activates a Mre11p-Tel1p G1/S Checkpoint in Budding Yeast

β-lapachone is an anticancer agent that selectively induces cell death in several human cancer cells. The mechanism of β-lapachone cytotoxicity is not yet fully understood. Here we report that β-lapachone treatment delayed cell cycle progression at the G1/S transition, incremented phosphorylation of the Rad53p checkpoint kinase and decreased cell survival in the budding yeast Saccharomyces cerevisiae. Furthermore, β-lapachone induced phosphorylation of histone H2A at serine 129. These checkpoint responses were regulated by Mec1p and Tel1p kinases. Mec1p was required for Rad53p/histone H2A phosphorylation and cell survival following β-lapachone treatment in asynchronous cultures, but not for the G1 delay. The tel1Δ mutation increased sensitivity to β-lapachone in a mec1 defective strain and compromised checkpoint responses in G1. Both Rad53p phosphorylation and G1 delay were fully dependent on a functional Mre11p-Rad50p-Xrs2p (XMR) complex, and mutants in the XMR complex were hypersensitive to β-lapachone treatment. Finally, XRS2 and TEL1 worked epistatically regarding β-lapachone sensitivity and Xrs2p was phosphorylated in a Tel1p dependent-manner after β-lapachone treatment. Taken together, these findings indicate that β-lapachone activates a Mre11p-Tel1p checkpoint pathway in budding yeast. Given the conserved nature of the Mre11p-Tel1p pathway, these results suggest that activation of the Mre11-Tel1p checkpoint could be of significance for β-lapachone antitumour activity.     

Phosphorylation of the axial element protein Hop1 by Mec1/Tel1 ensures meiotic interhomolog recombination

An essential feature of meiosis is interhomolog recombination whereby a significant fraction of the programmed meiotic double-strand breaks (DSBs) is repaired using an intact homologous non-sister chromatid rather than a sister. Involvement of Mec1 and Tel1, the budding yeast homologs of the mammalian ATR and ATM kinases, in meiotic interhomlog bias has been implicated, but the mechanism remains elusive. Here, we demonstrate that Mec1 and Tel1 promote meiotic interhomolog recombination by targeting the axial element protein Hop1. Without Mec1/Tel1 phosphorylation of Hop1, meiotic DSBs are rapidly repaired via a Dmc1-independent intersister repair pathway, resulting in diminished interhomolog crossing-over leading to spore lethality. We find that Mec1/Tel1-mediated phosphorylation of Hop1 is required for activation of Mek1, a meiotic paralogue of the DNA-damage effector kinase, Rad53p/CHK2. Thus, Hop1 is a meiosis-specific adaptor protein of the Mec1/Tel1 signaling pathway that ensures interhomolog recombination by preventing Dmc1-independent repair of meiotic DSBs.     

Transcription corepressor CtBP is an NAD(+)-regulated dehydrogenase

The Lcd1p/Mec1p complex is crucial for normal S phase progression and for signaling DNA damage. We show that Lcd1p/Ddc2p and Mec1p in cell extracts bind to DNA ends. Although Lcd1p binds DNA independently of Mec1p, recruitment of Mec1p to DNA requires Lcd1p. DNA binding by Lcd1p is also independent of Rad9p, Rad17p, and Rad24p. Recombinant Lcd1p binds DNA, and this is impaired by Lcd1p mutations that abrogate its in vivo functions. Furthermore, Mec1p is recruited to cdc13-induced DNA damage and HO endonuclease-induced double-strand breaks in vivo. This requires Lcd1p, and recruitment of Lcd1p/Mec1p to cdc13-induced damage is abolished by Lcd1p mutations that abrogate its in vivo functions. Recruitment of Lcd1p to these lesions is independent of Mec1p and Rad9p/Rad24p. Thus, recruitment of Mec1p to DNA lesions by Lcd1p is crucial for the DNA damage response.     

Hyperactivation of the yeast DNA damage checkpoint by TEL1 and DDC2 overexpression.

The evolutionarily conserved yeast Mec1 and Tel1 protein kinases, as well as the Mec1-interacting protein Ddc2, are involved in the DNA damage checkpoint response. We show that regulation of Tel1 and Ddc2-Mec1 activities is important to modulate both activation and termination of checkpoint-mediated cell cycle arrest. In fact, overproduction of either Tel1 or Ddc2 causes a prolonged cell cycle arrest and cell death in response to DNA damage, impairing the ability of cells to recover from checkpoint activation. This cell cycle arrest is independent of Mec1 in UV-irradiated Tel1-overproducing cells, while it is strictly Mec1 dependent in similarly treated DDC2-overexpressing cells. The Rad53 checkpoint kinase is instead required in both cases for cell cycle arrest, which correlates with its enhanced and persistent phosphorylation, suggesting that unscheduled Rad53 phosphorylation might prevent cells from re-entering the cell cycle after checkpoint activation. In addition, Tel1 overproduction results in transient nuclear division arrest and concomitant Rad53 phosphorylation in the absence of exogenous DNA damage independently of Mec1 and Ddc1.     

Mec1/ATR regulates the generation of single‐stranded DNA that attenuates Tel1/ATM signaling at DNA ends

Tel1/ATM and Mec1/ATR checkpoint kinases are activated by DNA double‐strand breaks (DSBs). Mec1/ATR recruitment to DSBs requires the formation of RPA‐coated single‐stranded DNA (ssDNA), which arises from 5′–3′ nucleolytic degradation (resection) of DNA ends. Here, we show that Saccharomyces cerevisiae Mec1 regulates resection of the DSB ends. The lack of Mec1 accelerates resection and reduces the loading to DSBs of the checkpoint protein Rad9, which is known to inhibit ssDNA generation. Extensive resection is instead inhibited by the Mec1‐ad mutant variant that increases the recruitment near the DSB of Rad9, which in turn blocks DSB resection by both Rad53‐dependent and Rad53‐independent mechanisms. The mec1‐ad resection defect leads to prolonged persistence at DSBs of the MRX complex that causes unscheduled Tel1 activation, which in turn impairs checkpoint switch off. Thus, Mec1 regulates the generation of ssDNA at DSBs, and this control is important to coordinate Mec1 and Tel1 signaling activities at these breaks.     

Rad53 is essential for a mitochondrial DNA inheritance checkpoint regulating G1 to S progression

The Chk2-mediated deoxyribonucleic acid (DNA) damage checkpoint pathway is important for mitochondrial DNA (mtDNA) maintenance. We show in this paper that mtDNA itself affects cell cycle progression. Saccharomyces cerevisiae rho(0) cells, which lack mtDNA, were defective in G1- to S-phase progression. Deletion of subunit Va of cytochrome c oxidase, inhibition of F(1)F(0) adenosine triphosphatase, or replacement of all mtDNA-encoded genes with noncoding DNA did not affect G1- to S-phase progression. Thus, the cell cycle progression defect in rho(0) cells is caused by loss of DNA within mitochondria and not loss of respiratory activity or mtDNA-encoded genes. Rad53p, the yeast Chk2 homologue, was required for inhibition of G1- to S-phase progression in rho(0) cells. Pif1p, a DNA helicase and Rad53p target, underwent Rad53p-dependent phosphorylation in rho(0) cells. Thus, loss of mtDNA activated an established checkpoint kinase that inhibited G1- to S-phase progression. These findings support the existence of a Rad53p-regulated checkpoint that regulates G1- to S-phase progression in response to loss of mtDNA.     

An enzyme in yeast mitochondria that catalyzes a step in branched-chain amino acid biosynthesis also functions in mitochondrial DNA stability.

The yeast mitochondrial high mobility group protein Abf2p is required, under certain growth conditions, for the maintenance of wild-type (rho+) mitochondrial DNA (mtDNA). We have identified a multicopy suppressor of the mtDNA instability phenotype of cells with a null allele of the ABF2 gene (delta abf2). The suppressor is a known gene, ILV5, encoding the mitochondrial protein, acetohydroxy acid reductoisomerase, which catalyzes a step in branched-chain amino acid biosynthesis. Efficient suppression occurs with just a 2- to 3-fold increase in ILV5 copy number. Moreover, in delta abf2 cells with a single copy of ILV5, changes in mtDNA stability correlate directly with changes in conditions that are known to affect ILV5 expression. Wild-type mtDNA is unstable in cells with an ILV5 null mutation (delta ilv5), leading to the production of mostly rho- petite mutants. The instability of rho+ mtDNA in delta ilv5 cells is not simply a consequence of a block in branched-chain amino acid biosynthesis, since mtDNA is stable in cells with a null allele of the ILV2 gene, which encodes another enzyme of that pathway. The most severe instability of rho+ mtDNA is observed in cells with null alleles of both ABF2 and ILV5. We suggest that ILV5 encodes a bifunctional protein required for branched-chain amino acid biosynthesis and for the maintenance of rho+ mtDNA.