Laboratories

Iwona J. Fijałkowska, PhD, DSc, Prof.

Laboratory of DNA Replication and Genome Stability

Position: Professor

ORCID: 0000-0002-5307-4332

E-mail:

Research Scope

Our laboratory seeks to identify mechanisms and factors that ensure efficient and highly accurate DNA replication and influence genetic stability.

Research

Main Scientific Achievements

  • Our data show that in S. cerevisiae, impaired GINS complex functioning may affect the division of labor providing circumstances under which DNA polymerase epsilon (Pol ε) is partially replaced on the leading strand by Pol δ
  • We demonstrated that S. cerevisiae CMGE dysfunctions result in genomic instability i.e. mutator phenotypes, defects in cell cycle progression, an increase in the participation of low-fidelity DNA Polζ, more frequent homologous recombination events, and an increase in the instability of repeat tracts
  • Our results show that in E. coli, the primary Ribonucleotide Excision Repair (RER) pathway dependent on RNase HII is more specific to the leading DNA strand, while RNase HI plays a bigger role in the lagging-strand RER. This bias is preserved during both normal replication and trans-lesion synthesis.
  • Based on our analyses of antimutator effect of the replicative DNA polymerase Pol III, we proposed that an increase in polymerase dissociation from terminal mismatches (i.e., potential mutations) is an important fidelity factor that can lead to mismatch removal.
  • We found that changes in the dNTP pool that are caused by deficiencies in the nucleoside diphosphate kinase and dCTP deaminase had a suppressive effect on constitutive SOS expression.

Research Description

Mechanisms that maintain high-fidelity DNA duplication and repair are critical to handle the malfunction of replication forks and DNA damage. Mutations are essential factors in long-term processes of evolution or the generation of antibody diversity, but in the short term they are deleterious and may cause human disease.

  • Escherichia coli research

By measuring the level of mutagenesis that occurs on leading and lagging DNA strands, we studied the involvement of particular DNA polymerases in the replication of both DNA strands and the mechanism of DNA polymerase switching1,2. In collaboration with R. Schaaper (NIEHS), we demonstrated that the accuracy of DNA replication is higher on the lagging strand3. This effect results from the more dissociative character of the lagging strand replication, which provides options for the activation of additional mutation-prevention pathways. Our conclusion is supported by results of antimutator polymerases that were characterized by an increase in dissociation rates4.

The E. coli SOS system is well-established as a model of the cellular response to DNA damage. In collaboration with R. Schaaper’s laboratory, we investigated the possible interplay between SOS and cellular dNTP pools. We found that alterations of the dNTP pool that were caused by deficiencies in the nucleoside diphosphate kinase and dCTP deaminase had a suppressive effect on constitutive SOS expression. We proposed a model of how alterations of the dNTP pool may interfere with the mechanism of SOS induction.

According to recent in vitro studies, DNA polymerases misincorporate rNTPs into DNA at a rate of 1 per 103 bp. Hence, ribonucleotides, which are more abundant in cells than dNTPs, vastly outnumber any other type of DNA damage or source of mutations. In collaboration with R. Woodgate (NIH), using steric gate mutants of DNA polymerases, we found that ribonucleotide incorporation and the efficiency of their subsequent removal were not equal on both DNA strands.

  • Bacteriophage RB69 research

The high fidelity of replication depends on polymerase nucleotide selectivity and the associated proof-reading activity of 3’-5’ exonuclease. To understand the molecular basis of base discrimination that is exhibited by replicative DNA polymerases, we studied DNA polymerase from bacteriophage RB69. RB69 polymerase, together with major eukaryotic DNA polymerases, belong to the B family. We conducted structure-function fidelity studies that allowed us to identify important residues that contribute to the high fidelity of this enzyme (Y567, S565)5, and we proposed that remote, non-catalytic residues may be critical for maintaining an optimal active site conformation (D714)6. We are also investigating interactions between the polymerase and primase from the T4 replisome.

  • Saccharomyces cerevisiae research

Studies of mutations of genes that encode replication complex proteins are important for understanding origins of human diseases. Replisome components are highly conserved in all eukaryotes. Therefore, yeast cells serve as an excellent model system to study their mutual functional dependencies. The central role in the replisome is played by the Cdc45-Mcm2-7-GINS (CMG) helicase complex, which also coordinates the assembly and function of replisome components. CMG associates with and stimulates the activity of DNA polymerase ε (Polε), forming the CMGE complex.

In our investigations, mutated variants of Polε or GINS of the CMGE complex impair genetic stability. We observed mutator phenotypes, defects in cell cycle progression, an increase in the participation of low-fidelity DNA Polζ, more frequent homologous recombination events, and an increase in the instability of repeat tracts7-10. Moreover, impaired functioning of the GINS complex provides circumstances under which DNA polymerase epsilon (Pol ε) is partially replaced by Pol δ on the leading strand11,12. We proposed that the defective function of CMGE impairs the progression of DNA replication, leading to a greater contribution of recombination-based repair mechanisms. These findings corroborate recent reports that defects in CMGE promote the development of certain human diseases.

To preserve genome integrity upon DNA replication perturbation, cell cycle checkpoint activation slows DNA synthesis, delays cell division, and activates a specific transcriptional program. Our studies of a mutant of the Dpb2 non-catalytic subunit of Polε demonstrated the involvement of Polε in sensing the proper progression of DNA replication on the leading DNA strand and activation of the transcriptional response to checkpoint activation8,13. This supports the model of parallel activation of the DNA replication checkpoint from the two DNA strands, in which Polε is involved in checkpoint activation from the leading strand.

  • Bibliography
  1. Fijalkowska IJ. et al. FEMS Microbiol Rev. 2012 doi: 10.1111/j.1574-6976.2012.00338.x
  2. Maliszewska-Tkaczyk M. et al. Proc Natl Acad Sci U S A. 2000 doi: 10.1073/pnas.220424697
  3. Fijalkowska IJ. et al.Proc Natl Acad Sci U S A. 1998 doi: 10.1073/pnas.95.17.10020
  4. Makiela-Dzbenska K. et al. DNA Repair (Amst). 2019 doi: 10.1016/j.dnarep.2019.102643
  5. Trzemecka A. et al. J Mol Biol. 2010. doi: 10.1016/j.jmb.2010.09.058
  6. Jacewicz A. et al. PLoS One. 2013. doi: 10.1371/journal.pone.0076700
  7. Garbacz M. et al. DNA Repair (Amst). 2015 doi: 10.1016/j.dnarep.2015.02.007
  8. Dmowski M. et al. PLoS Genet. 2017 doi: 10.1371/journal.pgen.1006572
  9. Jedrychowska M. et al. PLoS Genet. 2019. doi: 10.1371/journal.pgen.1008494
  10. Denkiewicz-Kruk M. et al. Int J Mol Sci. 2020. doi:10.3390/ijms21249484
  11. Dmowski M. et al. DNA Repair (Amst). 2022. doi:0.1016/j.dib.2022.108223
  12. Dmowski M. et al. Data in Brief. 2022 doi:0.1016/j.dib.2022.108223
  13. Dmowski M. et al. Curr Genet. 2017 doi: 10.1007/s00294-017-0706-7

Methodology

  • The chromosomal system for measuring the fidelity of leading and lagging DNA strand replication and participation of specific DNA polymerases in replication.
  • Alkaline gel electrophoresis to determine the amount of ribonucleotides in DNA.
  • Systems for measuring the instability of repeat tracts and recombination rates.
  • In vitro DNA replication fidelity assay.
  • Primer extension, DNA gel retardation, DNA binding, and exonuclease assays.

Selected Publications

  • Suppression of the coli SOS response by dNTP pool changes. Maslowska KH, Makiela-Dzbenska K, Fijalkowska IJ, Schaaper RM. Nucleic Acids Res. 2015. doi: 10.1093/nar/gkv217.
  • High-accuracy lagging-strand DNA replication mediated by DNA polymerase dissociation. Maslowska KH, Makiela-Dzbenska K, Mo JY, Fijalkowska IJ, Schaaper RM. Proc Natl Acad Sci U S A. 2018. doi: 10.1073/pnas.1720353115.
  • Defects in the GINS complex increase the instability of repetitive sequences via a recombination-dependent mechanism. Jedrychowska M, Denkiewicz-Kruk M, Alabrudzinska M, Skoneczna A, Jonczyk P, Dmowski M, Fijalkowska IJ. PLoS Genet. 2019. doi: 10.1371/journal.pgen.1008494.
  • Mutations in the Non-Catalytic Subunit Dpb2 of DNA Polymerase Epsilon Affect the Nrm1 Branch of the DNA Replication Checkpoint. Dmowski M, Rudzka J, Campbell JL, Jonczyk P, Fijałkowska IJ. PLoS Genet. 2017. doi: 10.1371/journal.pgen.1006572.
  • Increased contribution of DNA polymerase delta to the leading strand replication in yeast with an impaired CMG helicase complex. Dmowski M., Jedrychowska M., Makiela-Dzbenska K., Denkiewicz-Kruk M., Sharma S., Chabes A., Araki H., Fijalkowska I. J. DNA Repair (Amst). 2022. doi:10.1016/j.dnarep.2022.103272.

Collaborations

Prizes and Awards

  • Michal Dmowski. II° prize for published paper. Polish Genetics Society.

Publications (IBB PAS affiliated)

GRABOWSKA E., WROŃSKA U., DENKIEWICZ M., JASZCZUR M., RESPONDEK A., ALABRUDZIŃSKA M., SUSKI-GRABOWSKI C., MAKIELA-DZBEŃSKA K., JONCZYK P., FIJALKOWSKA I.J., Proper functioning of the GINS complex is important for the fidelity of DNA replication in yeast. Molecular Microbiology (2014) 92(4): 659-680 IF 5.026
GAWEL D., FIJALKOWSKA I.J., JONCZYK P., SCHAAPER R.M, Effect of dNTP pool alterations on fidelity of leading and lagging strand DNA replication in E. coli. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis (2014) 759: 22-28 IF 4.440
JACEWICZ A., TRZEMECKA A., GUJA K.E., PLOCHOCKA D., YAKUBOVSKAYA E., BEBENEK A., GARCIA-DIAZ M., A remote palm domain residue of RB69 DNA polymerase is critical for enzyme activity and influences the conformation of the active site. PLOS ONE (2013) 8(10): e76700 (14 p.) IF 3.730
FIJALKOWSKA I.J., SCHAAPER R.M, JONCZYK P., DNA replication fidelity in Escherichia coli: a multi-DNA polymerase affair. FEMS Microbiology Reviews (2012) 36(6): 1105-1121 IF 10.960
KRASZEWSKA J., GARBACZ M., JONCZYK P., FIJALKOWSKA I.J., JASZCZUR M., Defect of Dpb2p, a noncatalytic subunit of DNA polymerase ε, promotes error prone replication of undamaged chromosomal DNA in Saccharomyces cerevisiae. Mutation Research / Fundamental and Molecular Mechanisms of Mutagenesis (2012) 737(1-2) : 34-42 IF 2.850
MAKIELA-DZBEŃSKA K., JONCZYK P., SCHAAPER R.M, FIJALKOWSKA I.J., Proofreading deficiency of Pol I increases the levels of spontaneous rpoB mutations in E. coli. Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis (2011) 712: 28-32 IF 3.204
GAWEL D., JONCZYK P., FIJALKOWSKA I.J., SCHAAPER R.M, dnaX36 mutator of Escherichia coli : effect of the τ subunit of the DNA polymerase III holoenzyme on chromosomal DNA replication fidelity. Journal of Bacteriology (2011) 193(1): 296-300 IF 3.726
MAKIELA-DZBEŃSKA K., JASZCZUR M., BANACH-ORLOWSKA M., JONCZYK P., SCHAAPER R.M, FIJALKOWSKA I.J., Role of Escherichia coli DNA polymerase I in chromosomal DNA replication fidelity. Molecular Microbiology (2009) 74(5): 1114-1127 IF 5,213
JASZCZUR M., RUDZKA J., KRASZEWSKA J., FLIS K., POLACZEK P., CAMPBELL J.L., FIJALKOWSKA I.J., JONCZYK P., Defective interaction between Pol2p and Dpb2p, subunits of DNA polymerase epsilon, contributes to a mutator phenotype in Saccharomyces cerevisiae. Mutation Research / Fundamental and Molecular Mechanisms of Mutagenesis (2009) 669: 27-35 IF 3,198
GAWEL D., PHAM P.T., FIJALKOWSKA I.J., JONCZYK P., SCHAAPER R.M, Role of accessory DNA polymerases in DNA replication in Escherichia coli: analysis of the dnaX36 mutator mutant. Journal of Bacteriology (2008) 190(5): 1730-1742 IF 4,013
JASZCZUR M., FLIS K., RUDZKA J., KRASZEWSKA J., BUDD M.E., POLACZEK P., CAMPBELL J.L., JONCZYK P., FIJALKOWSKA I.J., Dpb2p, a voncatalyctic subunit of DNA polymerase ε, contributes to the fidelity of DNA replication in Saccharomyces cerevisiae. Genetics (2008) 178: 633-647 IF 4,001
JACEWICZ A., MAKIELA-DZBEŃSKA K., KIERZEK A.M., DRAKE J.W., BEBENEK A., The roles of Tyr391 and Tyr619 in RB69 DNA polymerase replication fidelity. Journal of Molecular Biology (2007) 368: 18-29 IF 4,89
SHCHERBAKOVA P.V., FIJALKOWSKA I.J., Translesion synthesis DNA polymerases and control of genome stability. Frontiers in Bioscience (2006) 11: 2496-2517 IF 2,771
KUBAN W., BANACH-ORLOWSKA M., SCHAAPER R.M, JONCZYK P., FIJALKOWSKA I.J., Role of DNA polymerase IV in Escherichia coli SOS mutator activity. Journal of Bacteriology (2006) 188: 7977-7980 IF 3,993
BANACH-ORLOWSKA M., FIJALKOWSKA I.J., SCHAAPER R.M, JONCZYK P., DNA polymerase II as a fidelity factor in chromosomal DNA synthesis in Escherichia coli. Molecular Microbiology (2005) 58: 61-70 IF 5,634
KUBAN W., BANACH-ORLOWSKA M., BIALOSKORSKA M., LIPOWSKA A., SCHAAPER R.M, JONCZYK P., FIJALKOWSKA I.J., Mutator phenotype resulting from DNA polymerase IV overproduction in Escherichia coli: preferential mutagenesis on the lagging strand. Journal of Bacteriology (2005) 187: 6862-6866 IF 3,993
KUBAN W., JONCZYK P., GAWEL D., MALANOWSKA K., SCHAAPER R.M, FIJALKOWSKA I.J., Role of Escherichia coli DNA polymerase IV in In Vivo replication fidelity. Journal of Bacteriology (2004) 186: 4802-4807 IF 4,146

Team

  • Iwona Fijałkowska, Prof., Head of Laboratory, ORCID: 0000-0002-5307-4332
  • Anna Bębenek, PhD, DSc, Employee, ORCID: 0000-0001-5374-8205
  • Michał Dmowski, PhD, Employee, ORCID: 0000-0002-6571-6259
  • Alina Krasilia, Employee
  • Krystian Łazowski, Employee, ORCID: 0000-0001-5163-7026
  • Karolina Makieła-Dzbeńska, PhD, Employee, ORCID: 0000-0002-1224-7444
  • Iwona Fijałkowska, Prof., ORCID: 0000-0002-5307-4332
  • Anna Bębenek, PhD, DSc, ORCID: 0000-0001-5374-8205
  • Karolina Makieła-Dzbeńska, PhD, ORCID: 0000-0002-1224-7444
  • Michał Dmowski, PhD, ORCID: 0000-0002-6571-6259
  • Milena Denkiewicz-Kruk, PhD, ORCID: 0000-0002-4357-9636
  • Krystian Łazowski, PhD student, ORCID: 0000-0001-5163-7026
  • Alina Krasilia, Technician

Grants

  • Differential activity of ribonucleotide repair on leading and lagging DNA strand and its consequences for the mechanism and fidelity of replication of both DNA strands in Escherichia coli cells. Krystian Łazowski. PRELUDIUM, National Science Center, 2020-2023
  • The CMGE helicase-polimerase complex a factor integrating the regulation of cell cyle progression and DNA replication. Michał Dmowski. HARMONIA, National Science Center, 2018-2023
  • The role of the Cdc45 protein in the maintenance of genomic stability in yeast Saccharomyces cerevisiae. Milena Denkiewicz-Kruk. PRELUDIUM, National Science Center, 2017-2023
  • Mechanisms of ribonucleotides incorporation and repair in E.coli cells. Karolina Makieła-Dzbeńska 2016-2021
  • Contribution of DNA polymerase delta in the leading strand replication in Saccharomyces cerevisiae cells. Iwona Fijałkowska. OPUS, National Science Center, 2016-2020
  • New players involved in the maintenance of genomic stability. Iwona Fijałkowska. TEAM, Foundation for Polish Science, 2012-2015