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Laboratory of Mutagenesis and DNA Repair

Head: Professor Iwona Fijałkowska

 


Regulation of fidelity of DNA replication in Escherichia coli and Saccharomyces cerevisiae cells

Group leader: Prof. Iwona J. Fijałkowska

Staff:
Senior Scientists:
Prof. Piotr Jonczyk, Dr Karolina Makieła-Dzbeńska, Dr Michał Dmowski
Graduate students: 
MSc Milena Denkiewicz-Kruk, MSc Małgorzata Jędrychowska, MSc Krystian Łazowski, MSc Ewa Szwajczak

 

The research conducted by the group of prof. Iwona Fijalkowska focuses on the important issue of how cells achieve high accuracy of transmission of genetic information. This issue of high fidelity is not only important for understanding the processes of long-term evolutionary change but also the ongoing development of mutant viruses and drug-resistant bacteria. Mutations occurring in human cells are also of critical importance, as they are causes of genetic diseases as well as somatic mutations.

The studies are focused on the fidelity mechanisms operating during DNA replication. The overall mutation rate (for virtually all organisms from bacteria to mammals) is low, in the range of only 10-9 to 10-11 mutations per nucleotide per round of replication, and how this high fidelity can be achieved is an important question.

While the fidelity of individual DNA polymerases, including their base insertion fidelity and proofreading ability, has been investigated in detail, recent emphasis has shifted to the fidelity of chromosomal multisubunit complexes (replisomes) that perform the simultaneous replication of leading and lagging DNA strands. Our group has been pursuing these issues in the prokaryotic (E. coli) and eukaryotic (S. cerevisiae) model systems using the genetic and biochemical approaches available for this organisms.

 

E.COLI RESEARCH


DNA replication fidelity in Escherichia coli: a multi-DNA polymerase affair

The unique experimental system that allows measuring of the level of mutagenesis occurring on leading and lagging DNA strands was developed in our laboratory. It was used to study the involvement of particular DNA polymerase in replication of leading and/or lagging DNA strand and mechanism of DNA polymerases switching during DNA replication. Our group, in collaboration with Roel Schaaper (NIEHS), demonstrated for the first time, that the accuracy of DNA replication is not equal for the two replicating DNA strands. Specifically, under normal conditions, replication of the lagging strand is more accurate than that of the leading strand. Based on our latest results we postulate that this effect results from the greater dissociative character of the lagging-strand polymerase, enabling activation of additional mutation-prevention pathways. Our conclusion is strongly supported by results obtained with dnaE antimutator polymerases characterized by increased dissociation rates.

These investigations allowed us to develop and present a scheme for E. coli DNA replication fidelity encompassing DNA polymerase competition and switching (Fig.1).

 

Suppression of the E. coli SOS response by dNTP pool changes

The E. coli SOS system is a well-established model for the cellular response to DNA damage. The control of SOS depends largely on the RecA protein. When RecA is activated by single-stranded DNA in the presence of a nucleotide triphosphate cofactor, it mediates cleavage of the LexA repressor, leading to expression of the 30+-member SOS regulon. RecA activation generally requires the introduction of DNA damage.  However, certain recA mutants, like recA730, bypass this requirement and display constitutive SOS expression as well as a spontaneous mutator effect. In collaboration with Roel Schaaper’s laboratory, we investigated the possible interaction between SOS and the cellular deoxynucleoside triphosphate (dNTP) pools.  We found that dNTP pool changes caused by deficiencies in the ndk or dcd genes, encoding nucleoside diphosphate kinase and dCTP deaminase, respectively, had a strongly suppressive effect on constitutive SOS expression in recA730 strains. The suppression of the recA730 mutator effect was alleviated in a lexA-deficient background. Overall, the findings suggest a model in which the dNTP alterations in the ndk and dcd strains interfere with the activation of RecA, thereby preventing LexA cleavage and SOS induction.

 

Mechanisms of ribonucleotides incorporation and repair in E.coli cells

 The ability of DNA polymerases to differentiate between ribonucleotides (rNTPs) and deoxribonucleotides (dNTPs) is fundamental for the accurate DNA replication and maintenance of genetic stability. 

 It is known that the intracellular concentration of rNTPs exceeds the levels of dNTPs by up to 2,000-fold. Recent in vitro experiments have demonstrated that DNA polymerases may misincorporate a ribonucleotide base every thousand base pairs, suggesting that rNTPs might be the most common non-canonical nucleotides inserted into genomic DNA. However, despite the high frequency of ribonucleotide incorporation in vitro, the level of misincorporated ribonucleotides persisting in genomic DNA in vivo is below current detection limits. This suggests that cells have evolved very efficient mechanisms for the removal of any erroneously incorporated rNMPs.

 To study the molecular mechanisms of the incorporation and removal of ribonucleotides in Escherichia coli cells in vivo, we are employing mutants in a steric gate residue of DNA polymerase V (isolated in Roger Woodgate’s laboratory). The steric-gate variants of DNA polymerases are excellent tools to study this topic as they enhance rNMPs incorporation and may also significantly alter the level of mutagenesis in the cell.

 The different physical properties of individual DNA polymerases e.g. the presence or absence of the 3'→5' exonuclease proofreading function, the ability to incorporate an incorrect nucleotide, and the ability to bypass errors in DNA, may influence the level of rNTPs incorporation and extension. We previously showed that participation of particular DNA polymerases during genome duplication differs between the leading and lagging DNA strand. Furthermore, during lagging strand replication RNA primers are frequently introduced and then removed, thus the participation of rNTPs in lagging strand synthesis is not the same as in the leading strand. Therefore, in collaboration with Roger Woodgate’s laboratory we are investigating whether the mechanisms related to rNTPs misincorporation and removal differ between the leading and lagging DNA strands. 

 

SACCHAROMYCES CEREVISIAE RESEARCH.

Beyond polymerases, DNA replication requires the coordinated action of a large number of other proteins. Thus, it is reasonable to ask whether these proteins do influence DNA replication fidelity and what may be the mechanism of their influence.

In eukaryotes, three B-family DNA polymerases, Pol alpha (Polα), Pol delta (Polδ) and Pol epsilon (Polε), are implicated in replication of chromosomal DNA in the S-phase of the cell cycle. Most of the major replicative DNA polymerases, often called replicases, consist of multi-subunit holoenzymes (HE). Although biochemical studies have provided insight into the role of the DNA polymerase catalytic subunits in the accuracy of DNA synthesis and organization of the holoenzymes, little is known about the role of accessory subunits in replication fidelity.

S. cerevisiae Polε, essential for leading strand DNA replication, are composed of four subunits encoded by the genes POL2, DPB2, DPB3 and DPB4. This composition of subunits is conserved from yeast to humans. Two subunits, Pol2p and Dpb2p, are essential in yeast. In order to investigate a possible role for the non-catalytic Dpb2p subunit in maintaining the fidelity of DNA replication, we isolated a series of temperature-sensitive mutants in the DPB2 gene. Newly isolated dpb2 alleles are strong mutators exhibiting mutation rates equivalent to pol2 mutants defective in the 3' → 5' proofreading exonuclease (pol2-4) or to mutants defective in mismatch repair (msh6).

Further results revealed impaired interaction between Pol2p and the mutator variants of Dpb2p. We observed an inverse correlation between the strength of the affinity between Dpb2p variants and Pol2p and the strength of the mutator phenotype that they conferred. We propose that the structural integrity of Pol ε holoenzyme is essential for accurate chromosomal DNA replication. We have also demonstrated that a destabilized DNA replicase holoenzyme enables much more frequent participation of Pol zeta (Polζ), a low-fidelity translesion DNA polymerase. The level of the partial Polζ-dependent spontaneous mutagenesis of dpb2 mutants suggests that the contribution of Polζ to genome replication in these mutants is higher than in wild-type strains.

 

 

Fidelity consequences of the impaired interaction between DNA polymerase epsilon (Polε) and the GINS complex

We have also shown that proper functioning of the GINS complex, a presumed platform synchronizing collaboration between replisome proteins in eukaryotic cells, is important for genetic stability in yeast cells. Based on the knowledge of the structure and the results from biochemical experiments, GINS was placed in the heart of the replisome as a novel factor essential for both the initiation and elongation stages of the DNA replication process. Biochemical analysis placed GINS as a component of the CMG [Cdc45-MCM helicase-GINS] complex and it was shown that GINS interacted directly with the major replicase DNA polymerase ε and with primase. Up to now the physiological data presenting biological function of GINS have been very limited. As the GINS complex is highly conserved in the archea and in all eukaryotes yeast cells serve as an excellent model system to study its functions in vivo.

Our data demonstrated that a mutant form of GINS causes spontaneous mutator phenotype, impaired interaction with Dpb2 subunit of DNA polymerase ε and increased participation of error-prone DNA polymerase ζ. An important outcome of this study is the demonstration that psf1 mutants exhibit a mutator phenotype, which indicates that defects in the GINS complex may positively correlate with genomic instability and the risk of diseases and carcinogenesis. This conclusion is in agreement with previously published data showing that GINS complex elements are upregulated in cancer cells and may serve as a prognostic biomarkers and that reduced expression of GINS complex proteins induces hallmarks of pre-malignancy in primary untransformed human cells.

 

Coordination of DNA replication and the cell cycle

To preserve genome integrity, the cell has to monitor DNA replication and coordinate it with cell cycle progression to avoid genomic instability. This is achieved through pathways known as cell cycle checkpoints whose role, upon DNA replication perturbation, is to slow down DNA synthesis, delay cell division and activate a specific transcriptional program. In response to the DNA replication stress, the cell maintains in the S phase elevated transcription of G1/S transition genes regulated by MBF and its co-repressor Nrm1. For that, through mechanisms that still need investigations, a specific signal is induced and propagated. Our studies of a mutant in DPB2, the gene coding for a non-catalytic subunit of Polε demonstrate the involvement of this DNA polymerase in (i) sensing proper progression of DNA replication on the leading DNA strand, and (ii) activation of the Nrm1-dependent (MBF) branch of the checkpoint response, which regulates the expression of many DNA replication checkpoint genes. Our results support the model of parallel activation of DNA replication checkpoint from the leading and lagging DNA strands. This strongly suggests that Polε, the leading strand replicase, is involved in DNA replication checkpoint activation from this strand. Our studies contribute to the understanding of mechanisms of cellular response to the DNA replication stress, which are necessary to preserve genome stability.

 

 

 

The ability of living cells to enter a new cell cycle and to control cell cycle progression is crucial for their viability and genetic stability. Eukaryotic cells are committed to undergo division before the onset of DNA replication, at the stage of G1 phase, called START in yeast, or restriction point in mammals. After that, cells proceed S, G2 and M phases following a tightly controlled program. The progression through the cell cycle is governed by kinases sequentially regulated by specific cyclins.

 

As the new cell cycle starts, expression of hundreds of genes (mainly regulated by the MBF and SBF transcriptional factors) are activated, peaks at the G1-S transition, and is inactivated in the S phase. We have shown that mutations in the gene coding for the essential non-catalytic Dpb2 subunit of DNA polymerase ε (Polε) result in perturbations the G1/S transition transcriptional program and in cell cycle progression. Polε, together with the GINS complex are components of the CMGE DNA helicase-polymerase complex and are recruited before the onset of DNA replication. Therefore, it might be speculated that Dpb2 is also involved in the regulatory processes during initiation of DNA replication.

 

SELECTED PUBLICATIONS:

 

Maslowska K. H., Makiela-Dzbenska K., Mo J-Y, Fijalkowska I. J., Schaaper R. M. (2018) High-accuracy lagging-strand DNA replication mediated by DNA polymerase dissociation. Proc. Natl. Acad. Sci USA. doi.org/10.1073/pnas.1720353115 

 

Szwajczak E, Fijalkowska IJ, Suski C. (2017) The importance of an interaction network for proper DNA polymerase zeta heterotetramer activity. Curr Genet. Nov 30. doi: 10.1007/s00294-017-0789-1. 

 

Szwajczak E, Fijalkowska I. J., Suski C. (2017) The CysB motif of Rev3p involved in the formation of the four-subunit DNA polymerase zeta is required for defective-replisome-induced mutagenesis, Molecular Microbiol. Sep 23. doi: 10.1111/mmi.13846.

 

Dmowski M., Fijałkowska I. J. (2017) Diverse roles of Dpb2, the non-catalytic subunit of DNA polymerase ε, Current Genetics 63:983-987.doi: 10.1007/s00294-017-0706-7

 

Dmowski M., Rudzka J., Campbell J. L., Jonczyk P., Fijałkowska I. J. (2017) Mutations in the Non-Catalytic Subunit Dpb2 of DNA Polymerase Epsilon Affect the Nrm1 Branch of the DNA Replication Checkpoint, PLOS Genetics 13:e1006572. doi: 10.1371/journal.pgen.1006572

 

Maslowska K.H., Makiela-Dzbenska K., Fijalkowska I.J., Schaaper R.M. (2015) Suppression of the E. coli SOS response by dNTP pool changes.Nucleic Acids Res. 43: 4109-4120. doi: 10.1093/nar/gkv217.

 

Garbacz M., Araki H., Flis K., Bebenek A., Zawada A.E., Jonczyk P., Makiela-Dzbenska K., Fijalkowska I.J. (2015) Fidelity consequences of the impaired interaction between DNA polymerase epsilon and the GINS complex. DNA Repair (Amst). 29: 23-35. doi: 10.1016/j.dnarep.2015.02.007.

 

Grabowska E., Wronska U., Denkiewicz M., Jaszczur M., Respondek A., Alabrudzinska M., Suski C., Makiela-Dzbenska K., Jonczyk P., Fijalkowska I.J. (2014) Proper functioning of the GINS complex is important for the fidelity of DNA replication in yeast. Mol Microbiol. 92: 659-680. doi: 10.1111/mmi.12580.

 

Gawel D, Fijalkowska I.J., Jonczyk P., Schaaper R.M. (2014)  Effect of dNTP pool alternations on fidelity of leading and lagging strand DNA replication in E. coli. Mutat Res. 759: 22-28. doi: 10.1016/j.mrfmmm.2013.11.003. 

 

Kraszewska J., Garbacz M., Jonczyk P., Fijalkowska I.J., Jaszczur M. (2012) Defect of Dpb2p, a noncatalytic subunit of DNA polymerase epsilon, promotes error prone replication of undamaged chromosomal DNA in Saccharomyces cerevisiae. Mutat Res. 737: 34-42. doi: 10.1016/j.mrfmmm.2012.06.002.

 

Fijalkowska I.J., Schaaper R.M., Jonczyk P. (2012) DNA replication fidelity in Escherichia coli: a multi-DNA polymerase affair. FEMS Microbiol Rev. 36: 1105-1121. doi: 10.1111/j.1574-6976.2012.00338.

 

 SELECTED PROJECTS:

 

(2018-2021) “CMGE helicase-DNA polymerase complex as a factor integrating the regulation of cell cycle and DNA replication” National Science Center “Harmonia” project for Michał Dmowski (IBB PAS, Poland) in collaboration with Hiroyuki Araki (National Institute of Genetics, Japan) and Etienne Schwob (Institut de Genetique Moleculaire de Montpellier, France).

 

(2016-2019) "Mechanisms of ribonucleotides incorporation and repair in E.coli cells." National Science Center 2015/18/M/NZ3/0040 “Harmonia” project for Karolina Makieła-Dzbeńska (IBB PAS, Poland) and Roger Woodgate (Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, USA).

 

(2017-2020) “The role of the Cdc45 protein in the maintenance of genomic stability in yeast Saccharomyces cerevisiae.” National Science Center 2016/21/N/NZ3/03255

 

(2016-2019) “Role of DNA polymerase delta in leading strand replication in S.cerevisiae cells.” National Science Center 2015/17/B/NZ1/00850  

 

(2012-2016) “The functional of non-catalytic Pol32 subunit of DNA polymerase delta at the replication fork” National Science Center 2011/03/B/NZ1/02773 

 

(2012-2015) “New players involved in the maintenance of genomic stability” TEAM/2011-8/1 Program of the Foundation for Polish Science and European Union Regional Development Fund

 

(2011-2015) "Studies of Nucleic Acids and Proteins - from Basic to Applied Research" MPD/2009-3/2 International PhD Projects Program of the Foundation for Polish Science and European Union Regional Development Fund

 

(2010-2015) Analysis of the fidelity of DNA synthesis of Pol epsilon in vitro - impact of noncatalytic Dpb2p subunit Ministry of Science and Higher Education, N N301 333439

 

 

 

Current staff:


Cellular responses of Saccharomyces cerevisiae to DNA damage

Group leader: Prof. Zygmunt Cieśla
Staff: Dr. Aneta Kaniak-Golik, Renata Kuberska, Prof. Ewa Śledziewska-Gójska
Collaboration: Dr. Piotr Dzierzbicki, Dr. Adrianna Skoneczna

 

The main line of research concerns mechanisms controlling the stability of mitochondrial DNA (mtDNA) in the yeast S. cerevisiae. The proximity of mtDNA to the electron transport chain makes it more vulnerable than nuclear DNA to damage by reactive oxygen species. If not repaired, lesions in mtDNA may result in mutations which are involved in pathogenesis of a variety of degenerative diseases, in cancer and in the normal aging process. The mechanisms responsible for the repair of mtDNA are poorly understood. It has been shown in this laboratory that the MSH1 gene, encoding mitochondrial homologue of the bacterial mismatch protein MutS, is involved in the prevention of oxidative lesions-induced mutagenesis. Our findings indicate that MutS plays a dual role in this process. On the one hand, Msh1p counteracts mitochondrial point mutagenesis induced by oxidative stress, and, on the other, Msh1p prevents rearrangements in the mitochondrial genome. Using a mitochondrial heteroallelic recombination assay, we have found that Msh1p has the capacity to promote allelic mitochondrial recombination. Since yeast genes involved in mitochondrial recombination are largely unknown, another goal of our studies  is to identify and characterize genes whose products are active at different steps of the mitochondrial recombination process. Within the research on this subject, we try to establish roles of mitochondrial nucleases Rad27 (the homolog of mammalian FEN1 nuclease), Nuc1 (the homolog of mammalian nucleases ENDOG and EXOG) and Din7 (the paralog of the yeast Exo1 nuclease) in mtDNA repair in normal conditions and in response to oxidative or other genotoxic stress. We analyze several aspects of repair and maintenance of the multicopy mitochondrial genome: integrity of wild-type genomes, point mutagenesis, mitochondrial homologous recombination and stability of microsatellite repeats. In our research, we include experiments using the pulsed-field electrophoresis method to follow changes in sizes and topologies of mtDNA molecules as well as changes in mtDNA copy number in yeast cells under oxidative stress. Studying the participation of the Rad27 nuclease, which has both mitochondrial and nuclear localization, in mtDNA repair, we found that cells lacking this nuclease exhibit several mitochondrial mutator phenotypes that do not result from the direct absence of Rad27 activity in the mitochondria, but rather arise due to indirect effects of Mec1/Rad53 kinase checkpoint pathway activation in response to the nuclear genome damage. Currently we search for regulatory mechanisms through which the conserved signaling pathway influences mtDNA stability. We also study mitochondrial functions of the Nuc1 nuclease. This conserved protein, known mostly for its role in an apoptotic pathway in yeast cells, in normal growth conditions is not localized to the mitochondrial matrix, where it may have access to mtDNA, but rather it is detected in the mitochondrial inter-membrane space. The protein is translocated into the matrix only in response to H2O2-induced oxidative stress. In addition to studies aimed at elucidating the functional significance of this oxidative-stress-induced Nuc1 translocation into the matrix, we try to pinpoint mitochondrial and non-mitochondrial signaling pathways that regulate this process.

 

Research grants (last 5 years):

 

2018-2021How activation of the Mec1/Tel1 kinase (human ATR/ATM) pathway in response to the nuclear genome damage influences the stability of mitochondrial genome: a study on the yeast model of mutations in polymerase POLG (National Science Centre)
2013-2018Maintenance of the mitochondrial genome stability in cells of model organism Saccharomyces cerevisiae: collaboration of numerous nucleases. (National Science Centre)
2010-2015 Mitochondrial homologous recombination in Saccharomyces cerevisiae cells: characterization of genes involved and proteins encoded by them in respect to their roles in the stability of mitochondrial genome (Ministry of Science and Higher Education)

 

Selected publications (last 5 years):

  1. Dzierzbicki P., Kaniak-Golik A., Malc E., Mieczkowski P., Cieśla Z. The generation of oxidative stress-induced rearrangements in Saccharomyces cerevisiae mtDNA is dependent on the Nuc1 (EndoG/ExoG) nuclease and is enhanced by inactivation of the MRX complex. Mutation Research / Fundamental and Molecular Mechanisms of Mutagenesis (2012) 740(1-2): 21-33
  2. Kaliszewska M., Kruszewski J., Kierdaszuk B., Kostera-Pruszczyk A., Nojszewska M., Łusakowska A., Vizueta J., Sabat D., Lutyk D., Lower M., Piekutowska-Abramczuk D., Kaniak-Golik A., Pronicka E., Kamińska A., Bartnik E., Golik P., Tońska K. Yeast model analysis of novel polymerase gamma variants found in patients with autosomal recessive mitochondrial disease. Human Genetics (2015) 134(9): 951-966 DOI: 10.1007/s00439-015-1578-x
  3. Kaniak-Golik A. and Skoneczna A. Mitochondria-nucleus network for genome stability. Free Radical Biology & Medicine (2015) 82: 73-104 DOI: 10.1016/j.freeradbiomed.2015.01.013
  4. Skoneczna A., Kaniak A., Skoneczny M. Genetic instability in budding and fission yeast – sources and mechanisms. FEMS Microbiology Reviews (2015) 39(6): 917-967 DOI: 10.1093/femsre/fuv028
  5. Kaniak-Golik A., Kuberska R., Dzierzbicki P., Śledziewska-Gójska E. Activation of Dun1 in response to nuclear DNA instability accounts for the increase in mitochondrial point mutations in Rad27/FEN1 deficient S. cerevisiae. PLOS ONE (2017) 12(7): e0180153 (30 p.) DOI: 10.1371/journal.pone.0180153.

 


Coordination of DNA repair processes in eukaryotes

 

Group leader: Prof. Ewa Śledziewska-Gójska

Staff:  Agnieszka Halas PhD, Dr. Justyna McIntyre PhD

Phd students: Michał Krawczyk MSc, Aleksandra Sobolewska MSc, Mikolaj Fedorowicz MSc

 

Up to 50,000 DNA lesions are generated every day in a single human cell. Part of these lesions escapes repair and persist in DNA until onset of DNA replication, which often (at least ones in each replication round) results in replication block.  On the other hand, faulty DNA replication results in chromosomal abnormalities leading to mutagenesis, carcinogenesis or cell death. Our group makes use of yeast and human cells to investigate the mechanisms involved in DNA damage tolerance (DDT) pathways, which allow to accomplish DNA replication, despite DNA damages blocking regular replicase complex. The main mechanism of DTT is translesion DNA synthesis (TLS) employing specialized TLS polymerases. Regulation of these enzymes is important because of their documented cancer suppression activities and, on the other hand, their negative influence on the action of a number of anticancer drugs. Our research focuses on regulation of cellular activity of Y family TLS polymerases: polymerase eta (Skoneczna et al., 2007; Plachta et al., 2015 [4]) and iota (McIntyre et al., 2013; 2015 [5]; 2015 [6]). Besides TLS, DTT employs mechanisms of homologous recombination. We analyze the factors coordinating activities of template switch and other homologous recombination pathways functioning in replication fork (Halas et al., 2011; 2016 [3]). Another project, we started recently, concerns the mechanisms controlling stability of mitochondrial DNA and a role of nuclear DNA replication stress in maintenance of this stability (Kaniak-Golik et al., 2017). In our investigations we cooperate with Dr Roger Woodgate from NICHS, NIH, Bethesda, USA (TLS polymerase iota project) and Dr Aneta Kaniak-Golik from IBB (mitochondrial DNA project).

 

Current projects:

  • Identification of new mechanisms engaged in DNA-damage tolerance pathways.
  • Factors regulating the levels and activities of TLS polymerases in yeast.
  • Regulation of human TLS polymerases eta and iota by post-tranlational modifications.
  • Mechanisms involved in regulation of genetic stability by homologous recombination.
  • The role of proteasome activity in modulation of genome stability.
  • Mechanisms of starvation-induced mutagenesis.
  • Mechanisms controlling stability of mitochondrial DNA.

Research projects:

2018-2021    "Promutagenic activity of ubiquine cinjugatin-like protein Mms2  in yeast S. cerevisiae.” (National Science Center)

2016-2019    "Regulation of posttranslational modification of human polymerase iota.” (National Science Centre)

2015-2017    "Cell cycle regulation of translesion DNA polymerase eta in response to UV radiation, in model cells of
                     Saccharomyces cerevisiae.” (National Science Center)

2013-2015    "Ubiquitination of human DNA repair polymerase iota.” (Foundation for Polish Sciences)

2011-2014    "DNA damage tolerance pathways: New mechanisms stimulating translesion synthesis.” (Ministry of Science and
                    Higher Education)

 

Selected publications (last 5 years):

  1. Lewandowski M, Kusiak MA, Michalczyk L, Szmigiel D, Sledziewska-Gojska E, Barzycka B, Wawrzyniak T, Luks T, Thordarson B, Wilde SA, Hoskuldsson A. (2017) Message in a stainless steel bottle thrown into deep geological time Gandvana Res. 52: 139-141
  2. Kaniak-Golik A, Kuberska R, Dzierzbicki P, Sledziewska-Gojska E. (2017) Activation of Dun1 in response to nuclear DNA instability accounts for the increase in mitochondrial point mutations in Rad27/FEN1 deficient S. cerevisiae. PLoS One. 12(7):e0180153.
  3. Halas A, Krawczyk M, Sledziewska-Gojska E. (2016) PCNA SUMOylation protects against PCNA polyubiquitination-mediated, Rad59-dependent, spontaneous, intrachromosomal gene conversion. Mutat Res. 791-792:10-18.
  4. Plachta M, Halas A, McIntyre J, Sledziewska-Gojska E. (2015) The steady-state level and stability of TLS polymerase eta are cell cycle dependent in the yeast S. cerevisiae. DNA Repair 29:147-53.
  5. McIntyre J, Woodgate R. Regulation of translesion DNA synthesis: Posttranslational modification of lysine residues in key proteins. (2015) DNA Repair 29:166-79.
  6. McIntyre J, McLenigan MP, Frank EG, Dai X, Yang W, Wang Y, Woodgate R. Posttranslational Regulation of Human DNA Polymerase ι. (2015) J Biol Chem. 290:27332-44.
  7. McIntyre J, Vidal AE, McLenigan MP, Bomar MG, Curti E, McDonald JP, Plosky BS, Ohashi E, Woodgate R. Ubiquitin mediates the physical and functional interaction between human DNA polymerases η and ι. (2013) Nucleic Acids Res. 41:1649-60.

 

  


Environmental stress as a source of genetic instability


Group leader: dr hab. Adrianna Skoneczna
Staff: PhD Kamil Król, M.Sc. Justyna Antoniuk-Majchrzak

Our team has been using the functional genomics methods to investigate the causes of genome instability of yeast Saccharomyces cerevisiae cells. Using a collection of yeast deletion mutants as well as Agilent’s platform based microarray technology allowed us to, among the others, to provide evidence for involvement of the vesicular transport in the maintenance of the genome stability. We proved that the vesicular transport role is not limited to the control of an uptake and detoxification of genotoxic compounds, but that the vesicular transport influence many pathways ensuring genome maintenance. These are the transduction of signal(s) about DNA damage, the activation of cellular defense mechanisms, the influencing the DNA damage repair and the exit from cell cycle arrest. Our studies allowed us to show that the cells of different ploidy react differently to the environmental stress and recruit dissimilar cellular mechanisms to prevent damage caused by these stresses. Additionally, we showed that the encountered stress may result not only in the appearance of the point mutations but may also lead to aneuploidy or even polyploidy. We also proved that the deletion of SWI6 gene is one of the factors involved in the above described phenomenon. We are especially interested in elucidation what mechanisms are involved in this process.

Current projects:

  • Identification of cellular mechanisms responsible for the maintenance of genome stability.
  • Studies on the mechanisms of the environmental stress response, especially to the genotoxic stress.
  • The contribution of the vesicular transport to genome maintenance.
  • Genetic and environmental factors affecting ploidy shift.
  • Swi6 involvement in regulation of transcription of the genes necessary for proper stress response.
  • The involvement of the Swi6 protein in the regulation of expression of genes indispensable for the appropriate cells’ response to stress.

Research:

2017-2020"New functional links in eukaryotic cell: Vesicular trafficking is a genome stability guardian", (National Science Centre)
2012-2016"Identification of Saccharomyces cerevisiae genes maintaining cell diploidy and genome stability", (National Science Centre)
2009-2011"Application of microarray technology in investigation of new genes responsible for DNA double strand breaks repair in yeast S. cerevisiae", (Ministry of Science and Higher Education)
2006-2009"Microarray approach towards identification yeast Saccharomyces cerevisiae genes engaged in genome stability", (Ministry of Science and Higher Education)

Selected publications:

 

  1. Krol K, Jendrysek J, Debski J, Skoneczny M, Kurlandzka A, Kaminska J, Dadlez M, Skoneczna A. (2017) Ribosomal DNA status inferred from DNA cloud assays and mass spectrometry identification of agarose-squeezed proteins interacting with chromatin (ASPIC-MS). Oncotarget. 8(15): 24988-25004.
  2. Natkańska U, Skoneczna A, Sieńko M, Skoneczny M. (2017) The budding yeast orthologue of Parkinson's disease-associated DJ-1 is a multi-stress response protein protecting cells against toxic glycolytic products. Biochim Biophys Acta. Biochim Biophys Acta. 1864(1): 39-50.
  3. Natkańska U, Skoneczna A, Sieńko M, Skoneczny M. (2016) The budding yeast orthologue of Parkinson's disease-associated DJ-1 is a multi-stress response protein protecting cells against toxic glycolytic products. Biochim Biophys Acta. doi: 10.1016/j.bbamcr.2016.10.016. [Epub ahead of print]
  4. Zadrag-Tecza R, Skoneczna A. (2016) Reproductive potential and instability of the rDNA region of the Saccharomyces cerevisiae yeast: Common or separate mechanisms of regulation? Exp Gerontol. 84:29-39. 
  5. Adamczyk J, Deregowska A, Skoneczny M, Skoneczna A, Natkanska U, Kwiatkowska A, Rawska E, Potocki L, Kuna E, Panek A, Lewinska A, Wnuk M. (2016) Copy number variations of genes involved in stress responses reflect the redox state and DNA damage in brewing yeasts. Cell Stress Chaperones. 21(5):849-64. 
  6. Adamczyk J, Deregowska A, Skoneczny M, Skoneczna A, Kwiatkowska A, Potocki L, Rawska E, Pabian S, Kaplan J, Lewinska A, Wnuk M. (2016) Adaptive response to chronic mild ethanol stress involves ROS, sirtuins and changes in chromosome dosage in wine yeasts. Oncotarget. 7(21):29958-76. 
  7. Skoneczna A, Kaniak A, Skoneczny M. (2015) Genetic instability in budding and fission yeast-sources and mechanisms. FEMS Microbiol Rev.39(6): 917-67.
  8. Krol K, Brózda I, Skoneczny M, Bretner M, Skoneczna A. (2015) A genomic screen revealing the importance of vesicular trafficking pathways in genome maintenance and protection against genotoxic stress in diploid Saccharomyces cerevisiae cells. PLOS One. 10(3): e0102-702.
  9. Kaniak-Golik A. i Skoneczna A. (2015) Mitochondria-Nucleus Network for Genome Stability. Free Radic. Biol. Med. 82: 73-104.
  10. Deregowska A, Adamczyk J, Kwiatkowska A, Gurgul A, Skoneczny M, Skoneczna A, Szmatola T, Jasielczuk I, Magda M, Rawska E, Pabian S, Panek A, Kaplan J, Lewinska A, Wnuk M. (2015) Shifts in rDNA levels act as a genome buffer promoting chromosome homeostasis. Cell Cycle. 14(21): 3475-87
  11. Deregowska A, Skoneczny M, Adamczyk J, Kwiatkowska A, Rawska E, Skoneczna A, Lewinska A, Wnuk M. (2015) Genome-wide array- CGH analysis reveals YRF1 gene copy number variation that modulates genetic stability in distillery yeasts. Oncotarget. 6(31): 30650-63.
  12. Alabrudzińska M, Skoneczny M, Skoneczna A. (2011) Diploid-specific genome stability genes of S. cerevisiae: Genomic screen reveals haploidization as an escape from persisting DNA rearrangement stress. PLOS One 6(6): e21124.
  13. Hałas A, Podlaska A, Derkacz J, McIntyre J, Skoneczna A, Śledziewska-Gójska E. (2011) The roles of PCNA SUMOylation, Mms2-Ubc13 and Rad5 in translesion DNA synthesis in Saccharomyces cerevisiae. Mol Microbiol. 80(3): 786-797.
  14. Malc E, Dzierzbicki P, Kaniak A, Skoneczna A, Cieśla Z (2009) Inactivation of the 20S proteasome maturase, Ump1p, leads to the instability of mtDNA in Saccharomyces cerevisiae. Mutat Res. 669(1-2): 95-103.
  15. Skoneczna A, McIntyre J, Skoneczny M, Policińska Z, Śledziewska-Gójska E. (2007) Polymerase eta is a short-lived, proteasomally degraded protein that is temporarily stabilized following UV irradiation in Saccharomyces cerevisiae. J. Mol. Biol. 366: 1074-1086.
  16. Skoneczna A, Miciałkiewicz A, Skoneczny M. (2007) Saccharomyces cerevisiae Hsp31p, a stress response protein conferring protection against reactive oxygen species. Free Radic. Biol. Med. 42: 1409-1420.
  17. McIntyre J, Baranowska H, Skoneczna A, Hałas A, Śledziewska-Gójska E. (2007) The spectrum of spontaneous mutations caused by deficiency in proteasome maturase Ump1 in Saccharomyces cerevisiae. Curr. Genet. 52: 221-228.