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Department of Molecular Biology

Head: Professor Jarosław Kuśmierek

The Department’s continuing interest is focused on the mechanisms of mutagenesis and DNA repair and different approaches are used to study this issue: (i) the response of E. coli cells to miscellaneous mutagens and the effects of various mutator and antimutator backgrounds on this response; (ii) the relation between structure of DNA bases adducts and their base-pairing and mutagenic/carcinogenic properties, as well as their repair; (iii) the structure-function relationship in fidelity of DNA polymerases. A considerable part of research is devoted to study of molecular basis of human disorders and includes studies on DNA damage in human tissues and on factors affecting its repair as potential factors of risk and progression of cancer disease and aging. Another part of research is the study of factors influencing microbiological homeostasis in the gastrointestinal tract of breeding animals. In addition, one project concerns construction of microbial fuel cells based on anaerobic microorganisms producing hydrogen and metal-reducing bacteria. 


Chemical basis of mutagenesis and repair of exocyclic base adducts

Group leader: Prof. Jarosław Kuśmierek

Staff: Prof. Jarosław Kuśmierek, Dr. Agnieszka Maciejewska, Dr. Beata Sokolowska, M.Sc. Karol Ruszel (Ph.D. student) 

 

Research
The research in laboratory is focused on the relationship between structure of DNA base adducts, mostly those formed during oxidative stress, i.e. generated by oxygen radicals and by products of lipid peroxidation, and their base-pairing, mutagenic and carcinogenic properties, as well as their repair. Current research activities: (i) degradation of mutagen-modified dNTPs by cellular enzymes, (ii) repair of exocyclic base adducts by DNA glycosylases, (iii) chloroacetaldehyde mutagenesis in E. coli, (iv) formation and structure of trans-4-hydroxy-2-nonenal adducts to DNA bases and (v) repair of exocyclic DNA base adducts by E. coli AlkB dioxygenase. 

 

Collaboration
Our research is done in collaboration with two other groups in our Department, leaded by Prof. Barbara Tudek and Prof. Elżbieta Grzesiuk. Out-of-institute collaboration: Prof. Ryszard Oliński, Nikolaus Copernicus University, Collegium Medicum in Bydgoszcz, Poland; Dr. Murat Saparbaev, Institute Gustave Roussy, Villejuif, France; Dr. Hilde Nilsen and Prof. Pal Falnes, University of Oslo, Norway.

Research grants:

2008-2010 "The AlkB protein and its eukaryotic homologues – the role in DNA repair and the possible role in cancer
                  etiology and target in cancer therapy" (Polish-Norwegian Research Found)

2006–2008 "Participation of AlkB protein in repair of ethenoadducts in DNA" (Ministry of Science and Higher Education)

 

Selected publications:

  1. Maciejewska A.M., Ruszel K.P., Nieminuszczy J., Lewicka J., Sokołowska B., Grzesiuk E., Kuśmierek J.T. Chloroacetaldehyde-induced mutagenesis in Escherichia coli: The role of AlkB protein in repair of 3,N(4)-ethenocytosine and 3,N(4)-α-hydroxyethanocytosine. Mutat. Res.-Fundam. Mol. Mech. Mut. (2010) 684: 24-34
  2. Komisarski M., Kaczmarska Z., Kusmierek J.T. Practical highly enantioselective synthesis of (R)- and (S)-(E)-4-hydroxynon-2-enal. Acta Biochim. Pol. (2009) 56: 189-193
  3. Maciejewska A.M., Jozwik A., Kusmierek J.T., Sokolowska B. Application of the k-NN classifier for mutagenesis tests. Recognition of wild type and defective in DNA repair bacterial strains on the basis of adaptive response to alkylating agents. Biocybern. Biomed. Eng. (2008) 28: 45-50
  4. Arczewska K.D., Michalickova K., Donaldson I.M., Nilsen H. The contribution of DNA base damage to human cancer is modulated by the base excision repair interaction network. Crit. Rev. Oncog. (2008) 14: 217-273
  5. Arczewska K.D., Kusmierek J.T. Bacterial DNA repair genes and their eukaryotic homologues: 2. Role of bacterial mutator gene homologues in human disease. Overview of nucleotide pool sanitization and mismatch repair systems. Acta Biochim. Polon. (2007) 54: 435-457
  6. Borys-Brzywczy E., Arczewska K.D., Saparbaev M., Hardeland U., Schar P., Kusmierek J.T. Mismatch dependent uracil/thymine-DNA glycosylases excise exocyclic hydroxyethano and hydroxypropano cytosine adducts. Acta Biochim Pol. (2005) 52: 149-165
  7. Speina E., Arczewska K.D., Gackowski D., Zielinska M., Siomek A., Kowalewski J., Olinski R., Tudek B., Kusmierek J.T. Contribution of hMTH1 to the maintenance of 8-oxoguanine levels in lung DNA of non-small-cell lung cancer patients. J. Natl. Cancer Inst. (2005) 97: 384-395
  8. Jurado J., Maciejewska A., Krwawicz J., Laval J., Saparbaev M.K. Role of mismatch-specific uracil-DNA glycosylase in repair of 3,N4-ethenocytosine in vivo. DNA Repair (Amst) (2004) 3: 1579-1590
  9. Maciejewska A.M., Lichota K.D., Kusmierek J.T. Neighbouring bases in template influence base-pairing of isoguanine. Biochem. J. (2003) 369: 611-618

Oxidative DNA damage

Group leader: Prof. Barbara Tudek

Staff: Dr. Elżbieta Speina, M.Sc. Alicja Winczura,  M.Sc. Beata Janowska, M.Sc. Paulina Prorok, M.Sc. Hubert Ludwiczak, Prof. Celina Janion (professor emeritus) 

 

Studies
Group is specialized in chemistry of DNA damage and enzymology of DNA repair. Main research areas include: mutagenic activity of oxidative DNA damage (8-oxoG, Fapy, exocyclic DNA adducts), repair pathways for DNA damage derived from lipid peroxidation products as well as the role of oxidative DNA damage repair in the ethiology of human cancers. Research includes clinical, and model studies performed on mammalian cell cultures, as well as in vitro experiments with purified repair proteins. Recently studies were extended to the mechanisms of aging, contribution of endogenous DNA damage and post-translational modification of repair proteins.


Collaborations
Our research is conducted in collaboration with numerous groups In Poland and abroad.  In IBB with groups headed by Prof. Jarosław Kuśmierek, Prof. Elżbieta Grzesiuk and Prof. Piotr Stępień. Collaboration with laboratories from outside IBB  in Poland: Prof. Ryszard Oliński, Prof. Małgorzata Zdzienicka, Collegium Medicum, Nicolaus Copernicus University,  Bydgoszcz, Prof. Joanna Rzeszowska-Wolny, Centerof Oncology, Gliwice, Prof. Hanna Radecka, Instytut Rozrodu Zwierząt i Badań Żywności, Olsztyn; abroad: Dr Murat Saparbaev, Dr Vasily Ogryzko, UMR 8126 C.N.R.S, University Paris-Sud, Institut Gustave-Roussy, Villejuif, France, Prof. Pal Falnes, Prof. Arne Klungland, Oslo University, Norway, Prof. Tinna Stevnsner, Aarhus University, Denmark, Prof. Vilhelm A. Bohr, National Institutes of Health, Baltimore, USA, Dr Wojciech Niedźwiedź, Oxford University, Great Britain

 

Research projects:

2008-2011 "Repair pathways of DNA damage induced by lipid peroxidation product trans-4-hydroxy-2-nonenal and their
                   role in genome stability and aging", (Ministry of Science and Higher Education)

2008-2011 "Repair of lipid peroxidation induced DNA damage: new proteins, new cancer risk factors and therapeutic
                   targets", (INCA - Ministry of Science and Higher Education)

2008-2011 "The AlkB protein and its eukaryotic homologues – the role in DNA repair and the possible role in cancer
                   etiology and target in cancer therapy", (Polish-Norwegian Research Fund)

2007-2009 "The role of lipid peroxidation in Werner syndrome", (Ministry of Science and Higher Education)

2006-2008 "Application of biosensors in molecular biology", (Ministry of Science and Higher Education)

2005-2009 "Studies on disorders in cellular signaling pathways in cancer pathogenesis with the use of integral genomic
                   methods investigated subtopic: Studies of oxidative processes in cancer pathogenesis with the use of integral
                   genomic methods", (Ministry of Science and Higher Education)

 

Selected publications:

  1. Langie S.A., Kowalczyk P., Tudek B., Zabielski R., Dziaman T., Oliński R., van Schooten F.J., Godschalk R.W. The effect of oxidative stress on nucleotide-excision repair in colon tissue of newborn piglets. Mutat Res. (2010) 695: 75-80
  2. Speina E., Dawut L., Hedayati M., Wang Z., May A., Schwendener S., Janscak P., Croteau D.L., Bohr V.A. Human RECQL5{beta} stimulates flap endonuclease 1. Nucleic Acids Res. (2010)
  3. Janowska B., Komisarski M., Prorok P., Sokołowska B., Kuśmierek J., Janion C., Tudek B. Nucleotide excision repair and recombination are engaged in repair of trans-4-hydroxy-2-nonenal adducts to DNA bases in Escherichia coli. Int. J. Biol. Sci. (2009) 5(6): 611-620
  4. Maddukuri L., Speina E., Christiansen M., Dudzińska D., Zaim J., Obtułowicz T., Kabaczyk S., Komisarski M., Bukowy Z., Szczegielniak J., Wójcik A., Kuśmierek J.T., Stevnsner T., Bohr V.A., Tudek B. Cockayne syndrome group B protein is engaged in processing of DNA adducts of lipid peroxidation product trans-4-hydroxy-2-nonenal. Mutat Res. (2009) 666(1-2): 23-31
  5. Bukowy Z., Harrigan J.A., Ramsden D.A., Tudek B., Bohr V.A., Stevnsner T. WRN Exonuclease activity is blocked by specific oxidatively induced base lesions positioned in either DNA strand. Nucleic Acids Res. (2008) 36(15): 4975-87
  6. Pereira M., Mason P., Szczesny R.J., Maddukuri L., Dziwura S., Jedrzejczak R., Paul E., Wójcik A., Dybczynska L., Tudek B., Bartnik E., Klysik J., Bohr V.A., Stepien P.P. Interaction of human SUV3 RNA/DNA helicase with BLM helicase; loss of the SUV3 gene results in mouse embryonic lethality. Mech. Ageing Dev. (2007) 128: 609-617
  7. Stobiecka M., Cieśla J.M., Janowska B., Tudek B., Radecka H. Piezoelectric sensor for determination of Genetically modified soybean roundup ready in samples not amplified by PCR. Sensors (2007) 7: 1462-1479
  8. Tudek B. Base excision repair modulation as a risk factor for human cancers. Mol. Aspects Med. (2007) 28: 258-275
  9. Speina E., Arczewska K.D., Gackowski D., Zielińska M., Siomek A., Kowalewski J.,  Oliński R., Tudek B., Kuśmierek J.T. Contribution of hMTH1 to the maintenance of 8-oxoguanine levels in lung DNA of non–small-cell lung cancer patients. J. Natl. Cancer Inst. (2005) 97: 384-395
  10. Speina E., Cieśla J.M., Grąziewicz J.-M., Laval J., Kazimierczuk Z., Tudek B. Inhibition of DNA repair glycosylases by base analogs and tryptophane pyrolisate, Trp-P1. Acta Biochim. Pol. (2005) 52: 167-178
  11. Gackowski D., Speina E., Zielińska M., Kowalewski J., Rozalski R., Siomek A., Paciorek T., Tudek B., Oliński R. Products of oxidative DNA damage and repair as possible biomarkers of susceptibility to lung cancer. Cancer Res. (2003) 63: 4899-4902
  12. Speina E., Zielińska M., Barbin A., Gackowski D., Kowalewski J., Grąziewicz M.-A., Siedlecki J.A., Oliński R., Tudek B.  Decreased repair activities of 1,N6-ethenoadenine and 3,N4-ethenocytosine in lung adenocarcinoma patients. Cancer Res. (2003) 63: 4351-4357

Induced and adaptive mutations

Group leader: Prof. Elżbieta Grzesiuk

Staff: Dr. Anna Sikora, Dr. Jadwiga Nieminuszczy, Dr. Joanna Krwawicz, M.Sc. Michał Wrzesiński, M.Sc. Damian Mielecki, M.Sc. Aleksandra Chojnacka  

 

Research
The team is studying mutagenesis and repair of damage to DNA in Escherichia coli. Specifically, we have investigated the role of AlkB dioxygenase in the repair of MMS-induced lesions in DNA. We have found that AlkB protein protects the cells not only against the genotoxic but also against the potent mutagenic activity of MMS by the repair of 1meA and 3meC lesions in DNA. MMS mutagenesis in alkB- mutants depends on the presence of PolV, the repair polymerase induced within SOS response, and the process of translesion synthesis, TLS. We have also studied the effect of the presence/absence of functional AlkB protein on MMS-induced mutagenesis and cell survival under conditions of exclusion of other DNA repair systems, BER, NER, TCR and MMR. We have found that AlkB protein is involved in the repair of apurinic/apirimidinic (AP) sites.

Separate project consists of investigation of antibacterial activity of pancreatic juice and microbiological homeostasis in the gastrointestinal tract of pigs.

We are also involved in the research on bacterial fermentations leading to hydrogen production and metagenomic analysis of selected consortia of hydrogen producing microorganisms. Dr. Anna Sikora is the leader of this group.
 

 

Collaboration
We collaborate with prof. Jarosław Kuśmierk (Departament of Molecular Biology IBB), prof. Romuald Zabielski (Department of Weterinary Medicine, Warsow University of Life Science) dr Urszula Zielenkiewicz (Departament of Biochemistry of Microorganisms IBB), prof. Mieczysław Błaszczyk (Agriculture Departament, Warsow University of Life Science) dr. Hilde Nielsen, prof. Pal Falnes and prof Arne Klungland (University of Oslo, Norvey).

Research grants:

2009-2011 "Metagenomic analysis of the selected hydrogen-producing consortium of fermentative bacteria cultivated
                   on organic waste products and analysis of the non-gaseous fermentation products", (Ministry of Sciences
                   and Higher Education)

2008-2011 "The AlkB protein and its eukaryotic homologues – the role in DNA repair and the possible role in cancer
                   etiology and target in cancer therapy", (Polish-Norwegian Research Fund)

2008-2011 "Functional analysis of At1g11780, At2g22260, At3g14160 and At5g01780 gene products in Arabidopsis thaliana
                   
- the structural homologues of bacterial AlkB protein", (Ministry of Sciences and Higher Education)

2006-2008 "The role of AlkB protein in the repair of etheno DNA adducts", (Ministry of Sciences and Higher Education)

2006-2008 "The involvement of AlkB protein In the repair of eteno adducts in DNA", (Ministry of Sciences and Higher
                   Education)

2005-2008 "Construction and optimization of microbiological fuel cells based on rumen microorganisms and metal reducing
                    bacteria", (Ministry of Sciences and Higher Education)

2004-2006 "Controlling the gastrointestinal tract development in animal neonatans to improve their survival and heath–
                   antybacterial activity of pancreatic juice", (Ministry of Sciences and Higher Education)

 

Selected publications:

  1. Wrzesiński M., Nieminuszczy J., Sikora A., Mielecki D., Chojnacka A., Kozłowski M., Krwawicz J., Grzesiuk E. Contribution of transcription-coupled DNA repair to MMS-induced mutagenesis in E. coli strains deficient in functional AlkB protein. Mutation Research (2010) - in press
  2. Sikora A., Mielecki D., Chojnacka A., Nieminuszczy J., Wrzesiński M., Grzesiuk E. Lethal and mutagenic properties of MMS-generated DNA lesions in Escherichia coli cells deficient in BER and AlkB-directed DNA repair. Mutagenesis (2009) doi:10.1093/mutage/gep052
  3. Maciejewska A.M., Ruszel K.P., Nieminuszczy J., Lewicka J., Sokołowska B., Grzesiuk E., Kuśmierek J.T.  Chloroacetaldehyde - induced mutagenesis in Escherichia coli: the role of AlkB protein in repair of 3,N(4)-ethenocytosine and 3,N(4)-alpha-hydroxyethanocytosine. Mutat Res. (2010) 684: 24-34
  4. Nieminuszczy J., Mielecki D., Sikora A., Wrzesiński M., Chojnacka A., Krwawicz J., Janion C., Grzesiuk E. Mutagenic potency of MMS-induced 1meA/3meC lesions in E. coli alkB strains. Environ Mol Mutagen (2009) 50: 791-799
  5. Sikora A., Chojnacka A., Zielenkiewicz U., Tomczyk K., Piela P., Grzesiuk E., Błaszczyk M.K. Hydrogen production by continuous culture of fermentative bacteria on waste substrates. Acta Microbiologica et Immunologica Hungarica (2009) 56: Suppl. p. 238
  6. Sikora A., Wójtowicz J., Kowalczyk P., Szewczyk K., Piela P., Chojnacka A., Grzesiuk E., Błaszczyk M.K. Continuous cultures of mixed populations of ferric (Fe+3) reducing bacteria. Acta Microbiologica et Immunologica Hungarica. (2009) 56: Suppl. p. 239
  7. Piela P., Michałowski T., Miltko R., Szewczyk K.W., Sikora R., Grzesiuk E., Sikora A. Electricity generation in a biohydrogen fuel cell using a fermentation gas produced by rumen ciliates. Acta Microbiologica et Immunologica Hungarica (2009) 56: Suppl. p. 226
  8. Jankowska A., Laubitz D., Wrzesinski M., Kazimierczak W., Skrzypek H., Bardowski J., Zabielski R., Grzesiuk E. Intestinal MMC-related electric fields and pancreatic juice control the adhesion of Gram-positive and Gram-negative bacteria to the gut epithelium - in vitro study. J. Physiol. Pharmacol. (2008) 59: 795-810
  9. Jankowska A., Laubitz D., Antushevich H., Zabielski R., Grzesiuk E. Competition of Lactobacillus paracasei with Salmonella enterica for adhesion to Caco-2 cells. J. Biomed. Biotech. (2008) ID: 357964, 6 pages
  10. Krwawicz J., Arczewska K., Speina E., Maciejewska A., Grzesiuk E. Bacterial DNA repair genes and their eukaryotic homologues: 1. Mutations in genes involved in base excision repair (BER) and DNA-end processors and their implication in mutagenesis and human disease. Acta Biochim. Pol. (2007) 54: 413-34
  11. Sikora A., Grzesiuk E. Heat shock response in gastrointestinal tract. J. Physiol. Pharmacol. (2007) 58 Suppl 3: 43-62
  12. Nieminuszczy J., Grzesiuk E. Bacterial DNA repair genes and their eukaryotic homologues: 3. AlkB dioxygenase and Ada methyltransferase in the direct repair of alkylated DNA. Acta Biochim. Polon. (2007) 54: 459-468
  13. Grzesiuk W., Nieminuszczy J., Kruszewski M., Iwanienko T., Płazińska M., Bogdańska M., Bar-Andziak E., Królicki L., Grzesiuk E. DNA damage and its repair in lymphocytes and thyroid nodule cells during radioiodine therapy in patients with hyperthyroidism. J. Mol. Endocrinol. (2006) 37: 527-32
  14. Laubitz D., Jankowska A., Sikora A., Woliński J., Zabielski R., Grzesiuk E. Gut myoelectrical activity induces heat shock proteins in Escherichia coli and Caco-2 cells. Experimental Physiology (2006) 91: 867-875
  15. Laubitz D., Lubańska A., Nieminuszczy J., Wrzesiński M., Jaworski A., Romanowicz K., Matyjek R., Grzesiuk E., Żebrowska T., Zabielski R. Pancreatic secretion of enzymes and antibacterial peptides differs according to the genotype of growing pigs. J. Physiol. Pharmacol. (2006) 57: 677-689
  16. Nieminuszczy J., Janion C., Grzesiuk E. Mutator specificity of Escherichia coli alkB117 allele. Acta Biochim. Polon. (2006) 53: 425-428
  17. Nieminuszczy J., Sikora A., Wrzesiński M., Janion C., Grzesiuk E. AlkB dioxygenese in preventing MMS-induced mutagenesis in Escherichia coli; effect of Pol V and AlkA proteins. DNA Repair (2006) 5: 181-188
  18. Wrzesiński M., Nowosielska A., Nieminuszczy J., Grzesiuk E. Effect of SOS-induced Pol II, Pol IV, and Pol V DNA polymerases on UV – induced mutagenesis and MFD repair in E.coli cells. Acta Biochim. Polon. (2005) 52: 139-147
  19. Nowosielska A., Wrzesiński M., Nieminuszczy J., Grzesiuk E. Mutator activity and specificity of Escherichia coli dnaQ49 allele - effect of umuDC products. Mutat Res. (2005) 572: 113-122

Mechanisms of DNA replication fidelity

Group leader: Dr. Anna Bębenek
Staff: M.Sc. Agata Jacewicz (Ph.D. student)

 

The current objective of our laboratory is to determine the mechanisms used by B family DNA polymerases in maintaining high fidelity of DNA replication. Our studies are focused on T even phage polymerase - RB69 DNA polymerase (gp43, a growth protein encoded by gene 43), a homologue of the genetically well characterized T4 DNA polymerase. We have selected RB69 DNA polymerase for several reasons: (a) this polymerase belongs to the B family of polymerases and shares a high degree of sequence similarity with eukaryotic replicative polymerases α, δ, and ε; (b) this enzyme works in concert with other accessory proteins to effect rapid and accurate genome replication of phage DNA in an analogous fashion to human replicases; (c) crystal structures are available for this polymerase, including the crystal structure for ternary complex (gp43, DNA primer-template, and incoming dNTP). This polymerase serves as a good model for structure-function studies.

The aims of our current projects are: (a) to establish the role of a weakly conserved palm-subdomain Asp714 residue in maintaining DNA replication; (b) we are looking for mutants that are defective in polymerase/exonuclease switching; (c) we survey the impact on fidelity of substitution at highly conserved residue S565, that is a part of a cluster of amino acids that form the rear wall of the nascent base-pair binding pocket, in primer extension and partitioning of the primer to the exonuclease site.

 

Collaboration
Our research is conducted in collaboration with dr John Drake from National Institute of Environmental Heath Sciences, NIH, Research Triangle Park, USA 

 

Research grants:

2007-2010 "Fidelity studies on RB69 DNA polymerase mutants DNA faga RB69", (Ministry of Sciences
                   and Higher Education)

2003-2007 "Fidelity studies on RB69 DNA polymerase mutants", (NIH/FIC )

2003-2006 "Fidelity studies on fingers and palm domain RB69 DNA polymerase mutants", (Ministry of Science
                   and Higer Education)

 

Selected publications:

  1. Trzemecka A., Płochocka D., Bebenek A. Different behaviours in vivo of mutations in the beta hairpin loop of the polymerases of the closely related phages T4 and RB69. J. Mol. Biol. 2009 389, 797-807.
  2. Bebenek A. Mechanizmy wierności replikacji DNA. Postępy Biochemii 2008 54: 43-56
  3. Jacewicz A., Makieła K., Kierzek A., Drake J.W., Bębenek A. The roles of Tyr391 and Tyr 619 in RB69 DNA polymerase replication fidelity. J. Mol. Biol. 2007 368: 18-29
  4. Zhang H., Rhee C., Bebenek A., Drake J.W., Wang J., Konigsberg W. The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Biochemistry 2006 45: 2211-2220
  5. Bebenek A., Carver G.T., Kadyrov F.A., Kissling G.E., Drake J.W. Processivity clamp gp45 and ssDNA-binding-protein gp32 modulate the fidelity of bacteriophage RB69 DNA polymerase in a sequence-specific manner, sometimes enhancing and sometimes compromising accuracy. Genetics. 2005 169:1815-1824
  6. Drake J.W., Bebenek A., Kissling G.E., Peddada S. Clusters of mutations from transient hypermutability. Proc.Natl. Acad. Sci. U.S.A 2005 102: 12849-12854
  7. Bebenek A., Carver G.T., Dressman H.K., Kadyrov F.A., Haseman J.K., Petrov V., Konigsberg W.H., Karam.J.D., Drake J.W. Dissecting the fidelity of bacteriophage RB69 DNA polymerase: site-specific modulation of fidelity by polymerase accessory proteins. Genetics 2002 162:1003-18