Kamila Kłosowska

Contribution of Base Excision Repair and AlkB-mediated direct reversal in the elimination of exocyclic adducts to DNA bases.

Inflammatory processes are involved in many human pathologies including certain types of cancers, e.g., colon, prostate and breast, so defense mechanisms are extremely important for individual susceptibility to cancer development, as well as for cancer treatment. Such an important defense mechanism is DNA repair. Lipid peroxidation-derived exocyclic DNA adducts, 1,N6-ethenoadenine (A), 3,N4-ethenocytosine (C), 1,N2-G and N2,3 -G, and bulky DNA adducts are among the major oxidative DNA base lesions with high miscoding potential, and thus important inducers of neoplastic transformation. A and C levels in DNA increase in oxidative stress-driven pro-cancerous diseases, like hemochromatosis, pancreatitis or ulcerative colitis (Bartsch and Nair, 2005). Ethenoadducts are removed from DNA by base excision repair (BER) pathway, initiated by N-methylpurine-DNA glycosylase (ANPG) for A, and mismatch-specific thymine-DNA glycosylase (TDG) for C (Gros et al., 2003). The exact repair pathway(s) for ethenoguanine adducts are not established. Recently, it was demonstrated that eA and eC can be also eliminated from DNA by oxidative dealkylation catalysed by AlkB protein, which removes alkyl groups by direct reversal without DNA scission and resynthesis (Delaney et al., 2005; Mishina et al., 2005). The exact enzymatic activities and cellular functions of this group of enzymes is very poorly elucidated, since it is not known to what extent they participate in the repair of exocyclic DNA adducts. Human cells contain at least eight homologues of bacterial AlkB protein. Their function is not limited to DNA repair, but they also participate in RNA modification, and some homologues seem to be engaged in signal transduction (Shimada et al.2009; Westbye et al., 2008.). Bacteria possess only one AlkB oxidoreductase that is expressed in E. coli as a part of the adaptive response-system to alkylating agents (Ada system) (Delaney et al., 2005). AlkB catalyses oxidation of cytotoxic alkyl groups at N1 of purines and N3 of pyrimidines in ssDNA, dsDNA and RNA, which leads to the reconstitution of intact DNA base (Sedwick et al., 2007). The major substrates for AlkB are 1meA and 3meC, but it repairs also 1meG, 3meT, as well as ethyl, propyl, hydroxypropyl and exocyclic DNA adducts formed during lipid peroxidation (1,N6-ethenoadenine and 3,N4-ethenocytsine) (Koivisto et al., 2003; Mishina et al., 2005). 1,N6-Ethenoadenine (A) and 3,N4-ethenocytsine (C) are repaired in E. coli also by BER glycosylases, AlkA glycosylase, which is also a component of Ada system and repairs A, and MUG DNA glycosylase, which excises from DNA C (Gros et al., 2003).

Objective of the study Objective of the study is to evaluate the contribution of Base Excision Repair and AlkB-mediated direct reversal in elimination from DNA exocyclic DNA adducts. Study will be performed in Escherichia coli, and if the desired cell lines will be available, may be extended to mammalian cells. IBB laboratory does not possess any cell lines defective in ANPG, TDG or any of ABH genes.

Project description Repair kinetics of A will be investigated in bacterial strains proficient and deficient either in BER or in AlkB or in both repair systems. Since bacteria generate very little, if any, ethenoadducts in their DNA, isolated plasmid DNA will be modified with ethenoadducts generating compound chloroacetaldehyde (CAA). These plasmids will be transformed into bacterial strains: wild type, alkA-, alkB- and the double mutant alkA- alkB-. Subsequently, bacteria will be grown in the presence of antibiotic maintaining the plasmid (e.g. ampicillin) and in the presence of a low concentration of alkylating agent, e.g. MMS in order to induce adaptive response. In different time intervals starting from transformation, plasmids will be re-isolated from bacteria, and ethenoadenine level will be estimated by the method(s) established in Dr Cook’s laboratory. Since AlkB protein may also be engaged in the processing of RNA, and hence affect repair of ethenoadducts by other systems, in parallel, a fraction of bacteria will be withdrawn during kinetic studies, and excision activity of A will be measured in cell lysates by the nicking assay in which radiolabeled oligodeoxynucleotide with a single A will be incubated with cell lysate. Following damaged base excision, oligodeoxynucleotide is cleaved with AP- endonuclease present in the lysate and the products will be separated by polyacrylamide electrophoresis. The intensity of bands is measured by phosphorimagering, and enzyme activity is estimated from the linear part of the curve. References: Bartsch H, Nair J (2005) Accumulation of lipid peroxidation-derived DNA lesions: Potential lead markers for chemoprevention of inflammation-driven malignancies. Mutat Res. 2005. Delaney, J.C., Smeester, L., Wong, C., Frick, L.E., Taghizadeh, K., Wishnok, J.S., Drennan, C.L., Samson, L.D. and Essigmann, J.M. (2005) AlkB reverses etheno DNA lesions caused by lipid oxidation in vitro and in vivo. Nat. Struct. Mol. Biol., 12, 855-860. Gros L, Ischenko AA, Ide H, Elder RH, Saparbaev MK (2003) Enzymology of repair of etheno-adducts. Mutat Res. 531:219-29. Koivisto P, Duncan T, Lindahl T, Sedgwick B. “Minimal methylated substrate and extended substrate range of Escherichia coli AlkB protein, a 1-methyladenine-DNA dioxygenase.” J Biol Chem. 2003 Nov 7;278(45):44348-54. Mishina Y, Yang CG, He C et al., Direct repair of the exocyclic DNA adduct 1,N6- ethenoadenine by the DNA repair AlkB proteins.” J Am Chem Soc. 2005 Oct 26;127(42):14594-5. Sedgwick B, Bates PA, Paik J, Jacobs SC, Lindahl T. “ Repair of alkylated DNA: recent advances.” DNA Repair. 2007 Apr 1;6(4):429-42. Shimada K, Nakamura M, Anai S, De Velasco M, Tanaka M, Tsujikawa K, Ouji Y, Konishi N. A novel human AlkB homologue, ALKBH8, contributes to human bladder cancer progression. Cancer Res. 2009 Apr 1;69(7):3157-64. Westbye MP, Feyzi E, Aas PA, Vågbø CB, Talstad VA, Kavli B, Hagen L, Sundheim O, Akbari M, Liabakk NB, Slupphaug G, Otterlei M, Krokan HE. Human AlkB homolog 1 is a mitochondria protein that demetylates 3-methylcytosine In DNA and RNA. J Biol Chem. 2008 Sep 5;283(36):25046-56