Pracownie badawcze

Badania

Najważniejsze osiągnięcia badawcze

• We described the roles of replication and DNA damage checkpoints in regulating the cellular abundance of Y family TS DNA polymerases in response to DNA metabolism insults.
• We discovered a new mechanism that controls the level of spontaneous mutagenesis, dependent on the noncanonical function of the ubiquitin-variant-E2 protein Mms2.
• We found that the polyubiquitination of PCNA stimulates Rad59-dependent homologous recombination.
• We uncovered the p300/CBP-dependent posttranslational regulation of DNA Pol iota by its acetylation and polyubiquitination.
• We found that upon activation by nuclear stress, the Mec1/Tel1-initiated kinase cascade indirectly influences mitochondrial DNA stability.

Opis badań

The integrity of genetic information is constantly threatened by DNA damage, which often remains unremoved before the start of DNA replication. The maintenance of genome functionality, despite the presence of DNA damage or defects in DNA replication machinery, depends on so-called DNA damage tolerance processes. These processes are conserved in all organisms and prevent genome rearrangements that are responsible for the acceleration of aging or can lead to cancer development and premature death.

The leading role in DNA damage tolerance is played by DNA polymerases (Pols), which are capable of translesion synthesis (TLS). Among TLS Pols, the main roles are played by the Y family Pols and Polζ, a B family member. For several years, we have investigated the regulation of the flag member of the Y family Pols, Polη. Polη is responsible for the tolerance of a number of DNA lesions, especially those that are caused by ultraviolet radiation, and its activity in human cells protects against 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, and the regulation of this enzyme is important. A method that was developed by our group for the detection of the native form of this Pol in yeast cells allowed us to establish that the cellular abundance of Polη is regulated in the cell cycle at the level of protein stability. We also found that under stress-free conditions, Polη accumulates mainly in the G2 phase, increasing the TLS potential of this cell cycle phase compared with the S phase. Our recent results indicated that this cell cycle regulation is modified in response to genotoxic stress-activating Mec1/Tel1-dependent checkpoint pathways. Activation of the DNA replication stress checkpoint (Mrc1) increases the level of Polη in the S phase. The growing activation of a DNA damage checkpoint (Rad9) progressively inhibits the accumulation of this Pol. We showed that this inhibition is common for all Y-family Pols in yeast. These results strongly suggest that damage tolerance mechanisms other than TLS are preferred in processing highly damaged DNA, whereas 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 orthologue, Polɩ. It is the most mutagenic of all known mammalian DNA Pols. The specific cellular function of Polɩ is still unknown. Our study of posttranslational Polɩ modifications revealed the role of Polɩ ubiquitination in the regulation of its interaction with other TLS factors. We also recently showed the contribution of p300/CBP acetyltransferase to the control of polyubiquitination, the stability of this enzyme, and its acetylation in the RIR domain in response to alkylating and oxidizing agents, which is unique to this TLS polymerase. We plan to identify 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 will also continue our attempts to determine the details of the process of Polɩ ubiquitination/deubiquitination.

Another TLS Pol that is the focus of our research is Polζ. The activity of this Pol is responsible for a part of spontaneous and the majority of DNA damage that is induced mutagenesis in eukaryotic cells. The lack of Polζ leads to embryonic death. We analyzed the functional consequences of a REV3L T2753R mutation that was identified in a retarded child with hypotrophy and dysmorphic features. This is the first point mutation that was identified in the gene that encodes the enzymatic subunit of Polζ with specific disease consequences in humans. Using a yeast model, we showed that an equivalent mutation increases the lethal effect of DNA-damaging agents and causes the instability of mitochondrial DNA.

Alternatively to TLS, DNA damage can be tolerated by damage avoidance via a template switch (TS). This process is initiated by polyubiquitination of the replication processivity factor PCNA by the Mms2-Ubc13-Rad5 complex. Our study showed that the activity of this complex is not limited to the TS. We found that it stimulates general gene conversion and that PCNA SUMOylation inhibits this recombination process. Additionally, our genetic studies suggested that the proteins of this complex play additional roles in controlling genetic stability. Our current research shows 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, Rsp5, and Def1 but not Ubc13 in the degradation of replicative Polδ, which leads to the more frequent replacement of this polymerase by error-prone Polζ and an increase in mutagenesis. Research on the mechanisms that are involved in this new process will continue.

Our research 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.

Metodologia

Our research employs standard techniques of molecular biology, biochemistry, genetics of yeast and yeast mitochondria, and tissue cultures. Our techniques include the construction of yeast strains (mating, cytoduction, gene replacement, and site-specific mutagenesis) and mammalian cell lines (Crispr-Cas9), yeast and yeast mitochondrial genome stability analysis (sensitivity to DNA-damaging agents, mutation rates, and frequency of homologous recombination), analyses of cell cycle progression (fluorescence-activated cell sorting), gene expression analysis (Northern blot, real-time reverse transcription polymerase chain reaction), and protein purification and analyses of their cellular and mitochondrial localization, stability, posttranslational modifications, and protein-protein interactions (yeast two-hybrid system, immunoprecipitation, pull-down, and far-Western).

Wybrane publikacje

• 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 S_N2 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
• Activation of Dun1 in response to nuclear DNA instability accounts for the increase in mitochondrial point mutations in Rad27/FEN1 deficient S. cerevisiae. Kaniak-Golik A, Kuberska R, Dzierzbicki P, Sledziewska-Gojska E. PLoS One. 2017 Jul 5;12(7):e0180153. doi: 10.1371/journal.pone.0180153
• The steady-state level and stability of TLS polymerase eta are cell cycle dependent in the yeast S. cerevisiae. Plachta M, Halas A, McIntyre J, Sledziewska-Gojska E. DNA Repair. 2015 doi: 10.1016/j.dnarep.2015.02.015

Współpraca

• 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/

Nagrody i wyróżnienia

• 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.