Laboratories

Research

Main Scientific Achievements

• We described the roles of replication and DNA damage checkpoints in regulating the cellular abundance of Y-family TLS DNA polymerases in response to DNA metabolism insults.
• We discovered a new mechanism controlling the level of spontaneous mutagenesis, dependent on the noncanonical function of the ubiquitin-variant E2 protein Mms2.
• We found that polyubiquitination of PCNA stimulates Rad59-dependent homologous recombination.
• We identified E3 ubiquitin ligase RNF2 and p300/CBP acetyltransferase, which on one hand interact with Polɩ, and on the other affect its cellular level in human cells.
• We found that yeast PCNA , a key protein in DNA replication and repair within nucleus, is localized to mitochondria and, upon genotoxic stress, can be involved in mitochondrial DNA translesion synthesis.

Research Description

The integrity of genetic information is constantly threatened by DNA damage, which often persists until DNA replication begins. The maintenance of genome functionality, even in the presence of DNA lesions or replication defects, depends on DNA damage tolerance (DDT) processes. These processes, conserved across all organisms, prevent genome rearrangements that accelerate aging or lead to cancer and premature death.

The central role in DNA damage tolerance is played by DNA polymerases (Pols), which are capable of translesion synthesis (TLS). Among TLS Pols, the Y-family members and Polζ (a B-family Pol) are key players. We have  long-lasting studies on the regulation of the flag member of the Y family Pols, Polη, which is responsible for tolerating several types of DNA lesions, especially those induced by ultraviolet radiation. The defect of Polη in human cells causes XP-V cancer predisposition. Polη (as well as other TLS Pols) can function in an error-prone manner. Both decreases and increases in Polη abundance cause genetic instability. Thus, its regulation is essential.

We developed a method to detect the native form of Polη in yeast cells, enabling us to show that its abundance is regulated via protein stability throughout the cell cycle. We also found that under non-stress conditions, Polη accumulates mainly in the G2 phase, increasing the TLS capacity of this cell cycle phase compared with the S phase. Our recent data show that this cell cycle regulation is modified in response to genotoxic stress via Mec1/Tel1-dependent checkpoint pathways.  Activation of the replication stress checkpoint (Mrc1) increases Polη levels in the S phase, while activation of the DNA damage checkpoint (Rad9) progressively inhibits Polη accumulation. We demonstrated that this inhibition affects all Y-family Pols in yeast. These findings strongly suggest that DNA damage tolerance mechanisms other than TLS are preferred in processing highly damaged DNA, while TLS preferentially functions in the S phase in response to defects of replication machinery or lower doses of DNA damage. Investigations of the mechanisms that are employed by checkpoint pathways to modulate DNA damage tolerance processes will be continued.

In human cells, in addition to Polη, there is also its closest paralogue, Polɩ. It is the most mutagenic of all known mammalian DNA polymerases; however,  despite the extensive characterization at the biochemical level, the specific cellular function of Polɩ remains unknown. The activity of Polɩ has been linked to various forms of cancer, underlining the necessity of the strict control of such error-prone enzyme. Protein interactions and posttranslational modifications play an important role in regulating Polι. Our study of posttranslational modifications of Polɩ (ubiquitination, acetylation) revealed their role in regulating Polɩ’s cellular level or its interaction with other TLS factors. We showed that p300/CBP acetylates Polɩ, and this modification is induced in response to alkylating and oxidizing agents. Additionally, p300/CBP acetyltransferase controls Polɩ’s polyubiquitination and its cellular level. Moreover, we recently identified RNF2, an E3 ubiquitin ligase, as a new interacting partner of Polι, whose activity is responsible for Polɩ stabilization. We work to identify different aspects of Polɩ function that are influenced by acetylation and explore the mechanisms that affect the cellular level of this enzyme, including the regulation of Polɩ expression under hypoxia conditions. We also continue our attempts to determine the details of the process of Polɩ ubiquitination/deubiquitination. Our goal is to identify the specific cellular function of Polɩ and decipher the mechanisms involved in its control.

Another TLS Pol of interest is Polζ, responsible for a part of spontaneous and the majority of DNA damage-induced mutagenesis in eukaryotic cells. Polζ deficiency leads to embryonic lethality. We investigated the functional consequences of a REV3L T2753R mutation, found in a child with hypotrophy and dysmorphic features—the first point mutation in the Polζ catalytic subunit with disease-specific consequences in humans. Using a yeast model, we showed that the equivalent mutation increases the lethal effect of DNA-damaging agents and causes the instability of mitochondrial DNA.

Aside from TLS, DNA damage can be bypassed by damage avoidance via a template switch (TS) mechanism, initiated by polyubiquitination of PCNA by the Mms2-Ubc13-Rad5 complex. Our studies revealed that the activity of this complex not only initiates TS but also promotes general gene conversion, which is inhibited by PCNA SUMOylation. Additionally, our genetic studies suggested that the proteins of this complex play additional roles in controlling genetic stability.

We recently uncovered a new, noncanonical function of Mms2 that is linked to a new pathway that controls spontaneous mutagenesis. We showed that Mms2, previously known only as a cofactor of Ubc13, cooperates with Ubc4 and Rsp5, but not Ubc13, in the degradation of replicative Polδ, which leads to the more frequent replacement of this polymerase by error-prone Polζ and results in an increased mutagenesis. Research on the mechanisms that are involved in this new process will continue.

Our recent work on mitochondria has shown that in response to nuclear DNA damage, the activation of a signal transduction pathway that is initiated by the conserved kinases Mec1 (human ATR) and Tel1 (human ATM) indirectly modulates the efficiency and accuracy of mtDNA replication. This previously unknown response of mitochondria to genotoxic stress and the role of TLS polymerases in this process are the subject of recently initiated studies by our group. We are also developing a study of a new and previously unrecognized function of Mec1/Tel1 kinases in the control of mitochondria. We have also recently shown that, surprisingly, yeast PCNA is localized to this compartment, and upon genotoxic stress, ubiquitinated PCNA is detected in highly purified mitochondria. The substitution K164R in PCNA leads to an increase of UV-induced point mutations in mtDNA, restricting entirely the anti-mutator replication bypass of UV damage in mtDNA by yeast Polη and partially also Polζ. Our other results suggest that PCNA participates in a mitochondrial replication damage bypass pathway dealing with lesions in DNA-RNA hybrids in mtDNA. Further studies will clarify the roles of TLS Pols in maintaining mitochondrial genome integrity under genotoxic stress.

Bibliography:

Latoszek et al., Sci Rep 2024. doi: 10.1038/s41598-024-82104-4.

Fedorowicz at al., BBA-Molecular Cell 2024. doi: 10.1016/j.bbamcr.2024.119743

Krawczyk et al. DNA Repair 2023. doi:10.1016/j.dnarep.2023.103484

Halas et al., J Mol Med. 2021 doi: 10.1007/s00109-020-02033-3

Halas et al., Curr Genet. 2020 doi: 10.1007/s00294-020-01061-3

McIntyre et al., Sci Rep.. 2019 doi: 10.1038/s41598-019-41249-3

Plachta M et al., DNA Repair. 2015 doi: 10.1016/j.dnarep.2015.02.015

Methodology

We use standard molecular biology, biochemistry, yeast genetics, mitochondrial research, and mammalian tissue culture techniques. These include:

• Yeast strain construction (mating, cytoduction, gene replacement, site-specific mutagenesis)
• CRISPR-Cas9 engineering of mammalian cell lines
• Genome stability assays in yeast, yeast mitochondria, and mammalian cells (sensitivity to DNA-damaging agents, mutation frequency, homologous recombination assays)
• Cell cycle analysis (FACS)
• Gene expression studies (Northern blotting, qRT-PCR)
• Protein purification and analysis of its subcellular localization, stability, and posttranslational modifications, as well as protein-protein interactions (yeast two-hybrid, immunoprecipitation, pull-down, far-Western).

Selected Publications

• PCNA and Rnh1 independently participate in the protection of mitochondrial genome against UV-induced mutagenesis in yeast cells. Latoszek M, Baginska-Drabiuk K, Sledziewska-Gojska E, Kaniak-Golik A. Sci Rep 2024. doi: 10.1038/s41598-024-82104-4.
• E3 ubiquitin ligase RNF2 protects polymerase ι from destabilization. Fedorowicz M, Halas A, Macias M, Sledziewska-Gojska E, Woodgate R, McIntyre J. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 2024. doi: 10.1016/j.bbamcr.2024.11974.
• Developmental delay with hypotrophy associated with homozygous functionally relevant REV3L variant. Halas A, Fijak-Miskal J, Kuberska R, Pienkowski VM, Kaniak-Golik A, Pollak A, Poznanski J, Rydzanicz M, Bik-Multanowski M, Sledziewska-Gojska E, Ploski R. J Mol Med. 2021 doi: 10.1007/s00109-020-02033-3.
• Regulation of the abundance of Y-family polymerases in the cell cycle of budding yeast in response to DNA damage. Sobolewska A, Halas A, Plachta M, McIntyre J, Sledziewska-Gojska E. Curr Genet. 2020 doi: 10.1007/s00294-020-01061-3.
• DNA polymerase ι is acetylated in response to SN2 alkylating agents. McIntyre J, Sobolewska A, Fedorowicz M, McLenigan MP, Macias M,Woodgate R, Sledziewska-Gojska E. Sci Rep.. 2019 doi: 10.1038/s41598-019-41249-3.

Collaborations

• Roger Woodgate, National Institute of Child Health and Human Development NIH, USA, https://www.nichd.nih.gov/research/atNICHD/Investigators/woodgate
• Rafał Ploski, Zakład Genetyki, Warsaw Medical University, PL https://genetyka.wum.edu.pl/content/zespol
• Matylda Macias, Laboratory of Molecular and Cellular Neurobiology, IIMCB, PL,https://www.iimcb.gov.pl/en/research/laboratories/5-laboratory-of-molecular-and-cellular-neurobiology
• Paweł Golik, Katarzyna Tońska, Institute of Genetics and Biotechnology, Warsaw University, PL https://www.igib.uw.edu.pl/index.php/start2/start/

Prizes and Awards

• Justyna McIntyre - HOMING PLUS Fellowship, 2013, Foundation for Polish Science, Poland.
• Mikołaj Fedorowicz - 1^st Prize, 2019, Science Slam Poland
• Mikołaj Fedorowicz - Prize for the Best Presentation, 2019, Forge of Young Talent, Academy of Young Scientists of the PAS.
• Mikołaj Fedorowicz - 2^ed Prize, 2018, FameLab Polska.