| 1. |
- Nicholls, Thomas J., et al.
(författare)
-
Dinucleotide Degradation by REXO2 Maintains Promoter Specificity in Mammalian Mitochondria
- 2019
-
Ingår i: Molecular Cell. - : Elsevier BV. - 1097-2765 .- 1097-4164. ; 76:5
-
Tidskriftsartikel (refereegranskat)abstract
- Oligoribonucleases are conserved enzymes that degrade short RNA molecules of up to 5 nt in length and are assumed to constitute the final stage of RNA turnover. Here we demonstrate that REXO2 is a specialized dinucleotide-degrading enzyme that shows no preference between RNA and DNA dinucleotide substrates. A heart- and skeletal-muscle-specific knockout mouse displays elevated dinucleotide levels and alterations in gene expression patterns indicative of aberrant dinucleotide-primed transcription initiation. We find that dinucleotides act as potent stimulators of mitochondrial transcription initiation in vitro. Our data demonstrate that increased levels of dinucleotides can be used to initiate transcription, leading to an increase in transcription levels from both mitochondrial promoters and other, nonspecific sequence elements in mitochondrial DNA. Efficient RNA turnover by REXO2 is thus required to maintain promoter specificity and proper regulation of transcription in mammalian mitochondria.
|
|
| 2. |
- Bacal, Julien, et al.
(författare)
-
Mrc1 and Rad9 cooperate to regulate initiation and elongation of DNA replication in response to DNA damage
- 2018
-
Ingår i: EMBO Journal. - : Wiley-VCH Verlagsgesellschaft. - 0261-4189 .- 1460-2075. ; 37:21
-
Tidskriftsartikel (refereegranskat)abstract
- The S-phase checkpoint maintains the integrity of the genome in response to DNA replication stress. In budding yeast, this pathway is initiated by Mec1 and is amplified through the activation of Rad53 by two checkpoint mediators: Mrc1 promotes Rad53 activation at stalled forks, and Rad9 is a general mediator of the DNA damage response. Here, we have investigated the interplay between Mrc1 and Rad9 in response to DNA damage and found that they control DNA replication through two distinct but complementary mechanisms. Mrc1 rapidly activates Rad53 at stalled forks and represses late-firing origins but is unable to maintain this repression over time. Rad9 takes over Mrc1 to maintain a continuous checkpoint signaling. Importantly, the Rad9-mediated activation of Rad53 slows down fork progression, supporting the view that the S-phase checkpoint controls both the initiation and the elongation of DNA replication in response to DNA damage. Together, these data indicate that Mrc1 and Rad9 play distinct functions that are important to ensure an optimal completion of S phase under replication stress conditions.
|
|
| 3. |
- Bru, Samuel, et al.
(författare)
-
Polyphosphate is involved in cell cycle progression and genomic stability in Saccharomyces cerevisiae
- 2016
-
Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 101:3, s. 367-380
-
Tidskriftsartikel (refereegranskat)abstract
- Polyphosphate (polyP) is a linear chain of up to hundreds of inorganic phosphate residues that is necessary for many physiological functions in all living organisms. In some bacteria, polyP supplies material to molecules such as DNA, thus playing an important role in biosynthetic processes in prokaryotes. In the present study, we set out to gain further insight into the role of polyP in eukaryotic cells. We observed that polyP amounts are cyclically regulated in Saccharomyces cerevisiae, and those mutants that cannot synthesise (vtc4 Delta) or hydrolyse polyP (ppn1 Delta, ppx1 Delta) present impaired cell cycle progression. Further analysis revealed that polyP mutants show delayed nucleotide production and increased genomic instability. Based on these findings, we concluded that polyP not only maintains intracellular phosphate concentrations in response to fluctuations in extracellular phosphate levels, but also muffles internal cyclic phosphate fluctuations, such as those produced by the sudden demand of phosphate to synthetize deoxynucleotides just before and during DNA duplication. We propose that the presence of polyP in eukaryotic cells is required for the timely and accurate duplication of DNA.
|
|
| 4. |
- Cohen, Rotem, et al.
(författare)
-
Ribonucleotide reductase from Fusarium oxysporum does not Respond to DNA replication stress
- 2019
-
Ingår i: DNA Repair. - : Elsevier. - 1568-7864 .- 1568-7856. ; 83
-
Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reductase (RNR) catalyzes the rate limiting step in dNTP biosynthesis and is tightly regulated at the transcription and activity levels. One of the best characterized responses of yeast to DNA damage is up-regulation of RNR transcription and activity and consequently, elevation of the dNTP pools. Hydroxyurea is a universal inhibitor of RNR that causes S phase arrest. It is used in the clinic to treat certain types of cancers. Here we studied the response of the fungal plant pathogen Fusarium oxysporum to hydroxyurea in order to generate hypotheses that can be used in the future in development of a new class of pesticides. F. oxysporum causes severe damage to more than 100 agricultural crops and specifically threatens banana cultivation world-wide. Although the recovery of F. oxysporum from transient hydroxyurea exposure was similar to the one of Saccharomyces cerevisiae, colony formation was strongly inhibited in F. oxysporum in comparison with S. cerevisiae. As expected, genomic and phosphoproteomic analyses of F. oxysporum conidia (spores) exposed to hydroxyurea showed hallmarks of DNA replication perturbation and activation of recombination. Unexpectedly and strikingly, RNR was not induced by either hydroxyurea or the DNA-damaging agent methyl methanesulfonate as determined at the RNA and protein levels. Consequently, dNTP concentrations were significantly reduced, even in response to a low dose of hydroxyurea. Methyl methanesulfonate treatment did not induce dNTP pools in F. oxysporum, in contrast to the response of RNR and dNTP pools to DNA damage and hydroxyurea in several tested organisms. Our results are important because the lack of a feedback mechanism to increase RNR expression in F. oxysporum is expected to sensitize the pathogen to a fungal-specific ribonucleotide inhibitor. The potential impact of our observations on F. oxysporum genome stability and genome evolution is discussed.
|
|
| 5. |
- Coquel, Flavie, et al.
(författare)
-
SAMHD1 acts at stalled replication forks to prevent interferon induction
- 2018
-
Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 557:7703, s. 57-61
-
Tidskriftsartikel (refereegranskat)abstract
- SAMHD1 was previously characterized as a dNTPase that protects cells from viral infections. Mutations in SAMHD1 are implicated in cancer development and in a severe congenital inflammatory disease known as Aicardi-Goutières syndrome. The mechanism by which SAMHD1 protects against cancer and chronic inflammation is unknown. Here we show that SAMHD1 promotes degradation of nascent DNA at stalled replication forks in human cell lines by stimulating the exonuclease activity of MRE11. This function activates the ATR-CHK1 checkpoint and allows the forks to restart replication. In SAMHD1-depleted cells, single-stranded DNA fragments are released from stalled forks and accumulate in the cytosol, where they activate the cGAS-STING pathway to induce expression of pro-inflammatory type I interferons. SAMHD1 is thus an important player in the replication stress response, which prevents chronic inflammation by limiting the release of single-stranded DNA from stalled replication forks.
|
|
| 6. |
- Gan, Haiyun, et al.
(författare)
-
Checkpoint Kinase Rad53 Couples Leading- and Lagging-Strand DNA Synthesis under Replication Stress
- 2017
-
Ingår i: Molecular Cell. - : Elsevier. - 1097-2765 .- 1097-4164. ; 68:2, s. 446-455
-
Tidskriftsartikel (refereegranskat)abstract
- The checkpoint kinase Rad53 is activated during replication stress to prevent fork collapse, an essential but poorly understood process. Here we show that Rad53 couples leading- and lagging-strand synthesis under replication stress. In rad53-1 cells stressed by dNTP depletion, the replicative DNA helicase, MCM, and the leading-strand DNA polymerase, Pol ε, move beyond the site of DNA synthesis, likely unwinding template DNA. Remarkably, DNA synthesis progresses further along the lagging strand than the leading strand, resulting in the exposure of long stretches of single-stranded leading-strand template. The asymmetric DNA synthesis in rad53-1 cells is suppressed by elevated levels of dNTPs in vivo, and the activity of Pol ε is compromised more than lagging-strand polymerase Pol δ at low dNTP concentrations in vitro. Therefore, we propose that Rad53 prevents the generation of excessive ssDNA under replication stress by coordinating DNA unwinding with synthesis of both strands.
|
|
| 7. |
- Garbacz, Marta A., et al.
(författare)
-
The absence of the catalytic domains of Saccharomyces cerevisiae DNA polymerase ϵ strongly reduces DNA replication fidelity
- 2019
-
Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 47:8, s. 3986-3995
-
Tidskriftsartikel (refereegranskat)abstract
- The four B-family DNA polymerases α, δ, ϵ and ζ cooperate to accurately replicate the eukaryotic nuclear genome. Here, we report that a Saccharomyces cerevisiae strain encoding the pol2-16 mutation that lacks Pol ϵ's polymerase and exonuclease activities has increased dNTP concentrations and an increased mutation rate at the CAN1 locus compared to wild type yeast. About half of this mutagenesis disappears upon deleting the REV3 gene encoding the catalytic subunit of Pol ζ. The remaining, still strong, mutator phenotype is synergistically elevated in an msh6Δ strain and has a mutation spectrum characteristic of mistakes made by Pol δ. The results support a model wherein slow-moving replication forks caused by the lack of Pol ϵ's catalytic domains result in greater involvement of mutagenic DNA synthesis by Pol ζ as well as diminished proofreading by Pol δ during replication.
|
|
| 8. |
- Jia, Shaodong, et al.
(författare)
-
Determination of deoxyribonucleoside triphosphate concentrations in yeast cells by strong anion-exchange high-performance liquid chromatography coupled with ultraviolet detection
- 2015
-
Ingår i: DNA Replikation. - New York : Springer-Verlag New York. - 9781493925957 - 9781493925964 ; , s. 113-121
-
Bokkapitel (refereegranskat)abstract
- DNA polymerase assays are commonly used for the detection of deoxyribonucleoside triphosphates (dNTPs) in biological samples. For better specificity and accuracy, high-performance liquid chromatography (HPLC) methods have been developed for the analysis of the four dNTPs in complex samples. Here we describe a simple method using isocratic strong anion-exchange (SAX) chromatographic separation coupled with ultraviolet detection (UV) for the analysis of the four dNTPs in budding yeast Saccharomyces cerevisiae. This method can be applied to other species of yeast or bacteria.
|
|
| 9. |
- Lanz, Michael Charles, et al.
(författare)
-
Separable roles for Mec1/ATR in genome maintenance, DNA replication, and checkpoint signaling
- 2018
-
Ingår i: Genes & Development. - : Cold Spring Harbor Laboratory. - 0890-9369 .- 1549-5477. ; 32:11-12, s. 822-835
-
Tidskriftsartikel (refereegranskat)abstract
- The Mec1/ATR kinase coordinates multiple cellular responses to replication stress. In addition to its canonical role in activating the checkpoint kinase Rad53, Mec1 also plays checkpoint-independent roles in genome maintenance that are not well understood. Here we used a combined genetic-phosphoproteomic approach to manipulate Mec1 activation and globally monitor Mec1 signaling, allowing us to delineate distinct checkpoint-independent modes of Mec1 action. Using cells in which endogenous Mec1 activators were genetically ablated, we found that expression of "free" Mec1 activation domains (MADs) can robustly activate Mec1 and rescue the severe DNA replication and growth defects of these cells back to wild-type levels. However, unlike the activation mediated by endogenous activator proteins, "free" MADs are unable to stimulate Mec1-mediated suppression of gross chromosomal rearrangements (GCRs), revealing that Mec1's role in genome maintenance is separable from a previously unappreciated proreplicative function. Both Mec1's functions in promoting replication and suppressing GCRs are independent of the downstream checkpoint kinases. Additionally, Mec1-dependent GCR suppression seems to require localized Mec1 action at DNA lesions, which correlates with the phosphorylation of activator-proximal substrates involved in homologous recombination-mediated DNA repair. These findings establish that Mec1 initiates checkpoint signaling, promotes DNA replication, and maintains genetic stability through distinct modes of action.
|
|
| 10. |
- Li, Shuqi, et al.
(författare)
-
Rtt105 functions as a chaperone for replication protein A to preserve genome stability
- 2018
-
Ingår i: EMBO Journal. - : Wiley-VCH Verlagsgesellschaft. - 0261-4189 .- 1460-2075. ; 37:17
-
Tidskriftsartikel (refereegranskat)abstract
- Generation of single-stranded DNA (ssDNA) is required for the template strand formation during DNA replication. Replication Protein A (RPA) is an ssDNA-binding protein essential for protecting ssDNA at replication forks in eukaryotic cells. While significant progress has been made in characterizing the role of the RPA-ssDNA complex, how RPA is loaded at replication forks remains poorly explored. Here, we show that the Saccharomyces cerevisiae protein regulator of Ty1 transposition 105 (Rtt105) binds RPA and helps load it at replication forks. Cells lacking Rtt105 exhibit a dramatic reduction in RPA loading at replication forks, compromised DNA synthesis under replication stress, and increased genome instability. Mechanistically, we show that Rtt105 mediates the RPA-importin interaction and also promotes RPA binding to ssDNA directly in vitro, but is not present in the final RPA-ssDNA complex. Single-molecule studies reveal that Rtt105 affects the binding mode of RPA to ssDNA These results support a model in which Rtt105 functions as an RPA chaperone that escorts RPA to the nucleus and facilitates its loading onto ssDNA at replication forks.
|
|
