Prof. dr hab. Iwona FijałkowskaPracownia Replikacji DNA i Stabilności Genomu
Our laboratory seeks to identify mechanisms and factors that ensure efficient and highly accurate DNA replication and influence genetic stability.
Najważniejsze osiągnięcia badawcze
- 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 (antimutator effect).
- 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.
- We showed that rNMPs that were inserted in both DNA strands by a steric gate mutant of polV were repaired in an RNase HII-dependent manner.
- We demonstrated that S. cerevisiae CMGE dysfunctions result in genomic instability.
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, and an increase in the instability of repeat tracts7-9. 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 activation10. 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.
- Fijalkowska IJ. et al. FEMS Microbiol Rev. 2012 doi: 10.1111/j.1574-6976.2012.00338.x
- Maliszewska-Tkaczyk M. et al. Proc Natl Acad Sci U S A. 2000 doi: 10.1073/pnas.220424697
- Fijalkowska IJ. et al.Proc Natl Acad Sci U S A. 1998 doi: 10.1073/pnas.95.17.10020
- Makiela-Dzbenska K. et al. DNA Repair (Amst). 2019 doi: 10.1016/j.dnarep.2019.102643
- Trzemecka A. et al. J Mol Biol. 2010. doi: 10.1016/j.jmb.2010.09.058
- Jacewicz A. et al. PLoS One. 2013. doi: 10.1371/journal.pone.0076700
- Garbacz M. et al. DNA Repair (Amst). 2015 doi: 10.1016/j.dnarep.2015.02.007
- Dmowski M. et al. PLoS Genet. 2017 doi: 10.1371/journal.pgen.1006572
- Jedrychowska M. et al. PLoS Genet. 2019. doi: 10.1371/journal.pgen.1008494
- Dmowski M. et al. Curr Genet. 2017 doi: 10.1007/s00294-017-0706-7
- 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.
- 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.
- Proper functioning of the GINS complex is important for the fidelity of DNA replication in yeast. Grabowska E, Wronska U, Denkiewicz M, Jaszczur M, Respondek A, Alabrudzinska M, Suski C, Makiela-Dzbenska K, Jonczyk P, Fijalkowska IJ. Mol Microbiol. 2014. doi: 10.1111/mmi.12580.
- Roel M. Schaaper Laboratory of Genome Integrity & Structural Biology, National Institute of Environmental Health Sciences, USA, https://irp.nih.gov/pi/roeland-schaaper
- Judith L. Campbell, Division of Biology and Biological Engineering, CALTECH, USA, https://www.bbe.caltech.edu/people/judith-l-campbell
- Hiroyuki Araki, Division of Microbial Genetics, National Institute of Genetics, Japan, https://www.nig.ac.jp/nig/research/interviews/faculty-interviews/hiroyuki-araki
- Roger Woodgate, Section on DNA Replication, Repair, and Mutagenesis, National Institute of Health, USA, https://www.nichd.nih.gov/research/atNICHD/Investigators/woodgate
- Etienne Schwob, Division of DNA Replication, Genome Instability & Cell Identity, Institute of Molecular Genetics, France, http://www.igmm.cnrs.fr/en/team/replication-et-instabilite-genomique/
- Adrianna Skoneczna, IBB PAS, Poland
- Izabela Kern-Zdanowicz, IBB PAS, Poland
Nagrody i wyróżnienia
- Michal Dmowski. II° prize for published paper. Polish Genetics Society.