Pracownie naukowe

Badania

Najważniejsze osiągnięcia badawcze

• We demonstrated that in Saccharomyces cerevisiae, CMGE (DNA helicase-polymerase) 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, an increase in the instability of repeat tracts, and 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 performed a multi-omics analysis of expression of the POLE2 non-catalytic subunit of DNA polymerase epsilon (Polε), showing its upregulation across multiple cancer types, what associates with decreased overall survival in a cancer-type-specific manner and modified transcriptional regulation involving E2F, EZH2, and RB.
• We have shown that during normal Escherichia coli replication, the fidelity of the lagging DNA strand is higher than that of the leading strand. We proposed that increased polymerase dissociation from terminal mismatches (i.e., potential mutations) is an important fidelity factor that can promote mismatch removal. The contribution of low-fidelity DNA polymerases may change this bias. Additionally, our results show that 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.
• We discovered that DNA polymerase ι (Pol ι), a highly mutagenic human translesion DNA polymerase, is acetylated by the p300/CBP acetyltransferases, and that inhibition of these enzymes induces Pol ι polyubiquitination and destabilization. We further identified the E3 ubiquitin ligase RNF2 as a regulator of Pol ι. Both p300/CBP and RNF2 interact with Pol ι and modulate its cellular abundance, revealing a potential mechanism by which acetylation and ubiquitination regulate the stability of this error-prone enzyme.
• We found that yeast PCNA, a key protein in DNA replication and repair within the nucleus, is localized to mitochondria and, upon genotoxic stress, can be involved in mitochondrial DNA translesion synthesis.

Opis badań

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 switching^1,2. 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 tracts^7-10. 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 strand^11,12. 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 activation^8,13. 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

Metodologia

• Bacterial and yeast chromosomal system for measuring the fidelity of leading and lagging DNA strand replication and participation of specific DNA polymerases in replication.
• Systems for measuring the instability of repeat tracts and recombination rates.
• In vitro DNA replication fidelity assay.
• Cell cycle analysis using flow cytometry
• Mammalian HPRT mutation assay;
• Real-time quantitative PCR (RT-qPCR) analysis
• In vitro acetylation and ubiquitination assays

Wybrane publikacje

• Krasilia A., Chaudhry D., Fijałkowska I.J., Dmowski M., Pan-cancer multi-omics analysis reveals upregulation of the POLE2 subunit of DNA polymerase epsilon and its associations with patient survival and cellular pathways beyond DNA replication. NAR Genomics and Bioinformatics (2026) 8(4): lqag118 (27 p.) doi.org/10.1093/nargab/lqag118
• Yazıcı E., McIntyre J. The complex network of p300/CBP regulation: Interactions, posttranslational modifications, and therapeutic implications. J Biol Chem. 2025. doi:10.1016/j.jbc.2025.110715
• Fedorowicz M, Halas A, Macias M, Sledziewska-Gojska E, Woodgate R, McIntyre J. E3 ubiquitin ligase RNF2 protects polymerase ι from destabilization. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 2024. doi:10.1016/j.bbamcr.2024.119743
• Latoszek M, Baginska-Drabiuk K, Sledziewska-Gojska E, Kaniak-Golik A. PCNA and Rnh1 independently participate in the protection of mitochondrial genome against UV-induced mutagenesis in yeast cells. Sci Rep 2024. doi: 10.1038/s41598-024-82104-4.
• Łazowski K., Faraz M., Vaisman A., Ashton N.W., Jonczyk P., Fijałkowska I.J., Clausen A.R., Woodgate R., Makieła-Dzbeńska K., Strand specificity of ribonucleotide excision repair in Escherichia coli. Nucleic Acids Research (2023) 51(4): 1766-1782 doi.org/10.1093/nar/gkad038

Współpraca

• Rosa Barrio and James D. Sutherland, Ubiquitin-likes and Development Lab, CIC bioGUNE, Spain, https://www.cicbiogune.es/people/rbarrio
• Damian Gaweł and Marlena Godlewska, Centre of Postgraduate Medical Education, Warsaw, Poland https://www.cmkp.edu.pl/ksztalcenie/szkola-nauk-medycznych/zaklad-biologii-komorki-i-immunologii/department-of-cell-biology-and-immunology-2
• Izabela Kern-Zdanowicz, IBB PAS, Poland
• Matylda Macias, Microscopy and Cytometry Facility, International Institute of Molecular and Cell Biology, https://mcf.iimcb.gov.pl/about-us/team/matylda-macias/
• Etienne Schwob, Division of DNA Replication, Genome Instability & Cell Identity, Institute of Molecular Genetics, France, https://www.igmm.cnrs.fr/en/team/replication-et-instabilite-genomique/
• Paweł Siedlecki, IBB PAS, Poland
• Adrianna Skoneczna, IBB PAS, Poland
• Roger Woodgate, Section on DNA Replication, Repair, and Mutagenesis, National Institute of Health, USA, https://www.nichd.nih.gov/research/atNICHD/Investigators/woodgate

Nagrody i wyróżnienia

• Łazowski K., Woodgate R., Fijałkowska I.J. - Prize for the best review paper published at IBB in 2024
• Łazowski K., Faraz M., Vaisman A., Ashton N.W., Jonczyk P., Fijalkowska I.J., Clausen A.R., Woodgate R., Makiela-Dzbenska K. - Prize for the best scientific paper published at IBB in 2023
• McIntyre J. - Prize for the best review paper published at IBB in 2020.
• Jędrychowska M., Denkiewicz-Kruk M. Alabrudziśka M., Skoneczna A., Jonczyk P., Dmowski M., Fijalkowska I.J. - Prize for the best scientific paper published at IBB in 2019