CURRICULUM VITAE SURNAME : FIRST NAME: Affiliation and official address: Date and place of birth: Nationality: Specialization ( specify)

Transcription

1 CURRICULUM VITAE SURNAME : Stepchenkova Affiliation and official address: FIRST NAME: Elena S.-Petersburg Branch of N.I. Vavilov Institute of General Genetics (Russian Academy of Sciences), Universitetskaya emb. 7/9, St., St. Petersburg, , Russia Date and place of birth: , Yartsevo, Russia Nationality: Russian Specialization (specify) (i) main field General and molecular genetics (ii) other fields Molecular biology, Microbiology (iii) current research interest Mutagenesis, Replication fidelity, Genetic toxicology Education (degrees, dates, universities) Bachelor of Biology, 1999, St.Petersburg State University, St. Petersburg, Russia, Master in science of Biology, 2001, St.Petersburg State University, St. Petersburg, Russia, Graduate student, , St.Petersburg State University, St. Petersburg, Russia, PhD, 2006, St.Petersburg State University, St. Petersburg, Russia Career/Employment (employers, positions and dates) Laboratory Assistant of the staff in the Biological Institute, St. Petersburg State University, 2003 present Junior staff scientist at the Laboratory of Physiological Genetics, Saint-Petersburg State University, 2005 present Junior staff scientist at the S.-Petersburg Branch of N.I. Vavilov Institute of General Genetics (Russian Academy of Sciences), (with NATO CLG support); (with NATO CLG support) and Worked as a special volunteer in the laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NC, USA, Worked as a special volunteer in University of Nebraska Medical Center, Omaha, NE, USA. Teaching 2007 A 14 hours lecture course Replication and mutagenesis was developed and conducted for Master program students at department of Genetics and breeding, Saint Petersburg State University

2 PUBLICATIONS: - Number of papers in refereed journals: 1. Stepchenkova E. I., Kozmin S. G., Alenin V. V., Pavlov Y. I. Genome-wide screening for genes whose deletions confer sensitivity to mutagenic purine base analogs in yeast // BMC Genetics : Kochenova O.V., Borhsenius A.S., Stepchenkova E.I., Inge-Vechtomov S.G. Genetic control of matting type in yeast Saccharomyces cerevisiae as a base for development of test-system (alpha-test) to estimate genetic activity of endogenous and exogenous agents. // Complied works of the Biological Institute. 54 «Fundamental bases of innovative biological projects in «Naukograd», Russian 3. Stepchenkova E. I., Kozmin S. G., Alenin V. V., Pavlov Y. I. Genetic control of metabolism of mutagenic base analogs 6-hydroxylaminopurime and 2-amino-6- hydroxylaminopurime in yeast Saccharomyces cerevisiae // Genetika. V In press - Number of communications to scientific meetings: more then 10 Conferences attended in 2008: 1. Kochenova O.V., Stepchenkova E.I., Pavlov Y.I., Shsherbakova P.V., Inge- Vechtomov S.G. The α-test, DNA-polymerase zeta and stability of genetic material. In: the summary of international school-conference Problems of bioecology and the ways of its probable solving (II Rzhavitinskii readings), Saransk (Abstract) 2. Menezes M., Spitler K, Stepchenkova E., Borgstahl G. Pavlov Y. Structure and Function of human ITPA, a nucleotide pool sanitizing enzyme // 10 th Annual Midwest DNA Repair Symposium. Pittsburgh, PA; USA (Abstract). 3. Stepchenkova E.I., Kochenova O.V., Pavlov Y.I., Shcherbakova P.V., Inge- Vechtomov S.G. Detection of 8-oxoguanine primary lesions in selective system of illegitimate cytoduction.//10 th Annual Midwest DNA Repair Symposium. Pittsburgh, PA; USA (Abstract) 4. Menezes M., Stepchenkova E., Spitler K, Borgstahl G. Pavlov Y. Structure and Function of human ITPA, a nucleotide pool sanitizing enzyme // Gordon Research conference. Mutagenesis. Oxford. England. July 20-25, (Abstract) 5. Коченова О.В. Степченкова Е.И. Тест-система для анализа генетической активности экзогенных и эндогенных факторов. //Одиннадцатая Всероссийская медико-биологическая конференция молодых исследователей «Человек и его здоровье».фундаментальная наука и клиническая медицина, Санкт-Петербург, Inge-Vechtomov S.G., Stepchenkova E.I., Kochenova O.V., Shumega A.R., Zadorsky S.P., Shcherbakova P.V. Alpha-test in yeast Saccharomyces cerevisiae for genetic toxicology and fundamental research. 12th International Congress on Yeasts. Kiev, Ukraine August 11-15, 2008.

3 Review of PhD thesis by Elena Stepchenkova Genetic control of sensitivity to mutagenic analogs of purine bases in pro- and eukaryotic microorganisms Sanitization of the cellular nucleotide precursor pools of mutagenic base analogs is required for high fidelity replication of genetic material. The process of nucleotide pool sanitization reduces the level endogenous mutagenesis. This, in turn, decreases the onset of aging as well as diseases like cancer which are dependent on mutagenic events for their initiation. The undesirable mutagenic/recombinogenic/toxic incorporation of purine base analogs into nucleic acids (e.g. ITP, ditp or 6-hydroxyaminopurine deoxynucleoside triphosphate (dhaptp) is prevented by a putative oncogene, inosine triphosphate pyrophosphatase (ITPA). dhaptp is a synthetic adenine analog that is highly mutagenic because it is incorporated by DNA polymerases opposite either a T or a C. For the first time in the limits of dissertation topic we carried out a genome wide screen for genes conferring sensitivity to mutagenic base analogs in yeast S. cerevisiae and bacterium E. coli. Using this method we studied the role of all nonessential genes in controlling cell sensitivity to HAP. Some of genes, which were identified in genomewide screen also control sensitivity to 2-amino-HAP (AHAP) and hypoxantine. Our results let us to make following conclusions: 1. Using modern methods of functional genomics we have identified 20 genes in yeast S. сerevisiae and more then 40 genes in bacteria E. coli whose inactivation leads to change in sensitivity to HAP. 2. Beside HAP sensitivity yeast mutants aah1 and yjl055w shoved sensitivity to another base analog AHAP. HAP-sensetive mutants ade12 and ade13 are sensitive to AHAP and natural precursor of adenine and guanine hypoxanthine. 3. In bacteria and yeast HAP detoxification pathway utilizes different mechanisms. In bacteria molybdenum cofactor dependent system plays the main role in protection from HAP. In yeast main players in this process are purine salvage enzymes encoded by the НАМ1, ADE12 и ADE13 genes. In yeast an opposite of bacterial situation take a place. Inactivation of any gene

4 from the purine biosynthesis de novo pathway leads to elevated HAP sensitivity. 4. HAP-induced mutagenesis either in yeast or bacteria does not depend on general repair systems. 5. The common feature of the HAP protecting systems in yeast and bacteria is requirement of purine salvage enzymes. 6. For activation of HAP as a mutagen in both studied species activity of phosphorybisiltransferases is required. On the base of our results we made a conclusion that at least three different systems for protection from HAP exist in model microorganisms. There are human homologues of the genes, which were identified in the genome-wide screen. Several human heritable diseases are caused by inactivation of those genes. Yeast and bacterial strains, carrying mutations of the corresponding genes, may serve as a model for those disorders research. Results presented in the applicant s PhD thesis may be useful for creation of new test-systems in genetic toxicology.

5 Short review of present research conducted by Elena Stepchenkova In field of Mutagenesis During last two years after PhD thesis defense our work has been developed in two main directions. The first one is devoted to study of DNA precursor pool sanitation mechanisms. Presence of some noncanonical base analogs in the pool of DNA precursors decreases DNA replication fidelity. Mutagenic base analogs may cause different heritable diseases and cancer that determinates all importance of detailed research of the base analogs- induced mutagenesis. The simplest eukaryotic organism yeast S. cerevisiae is a very useful model to study molecular mechanism of base analog-induced mutagenesis. In the present project we are utilizing all advantages of yeast model to study genes from other organisms, which are predicted to be important for control of sensitivity to base analog 6-hydroxylaminopurine (HAP) (Stepchenkova et al., 2005; Stepchenkova at al., 2009). With this purpose we are studying genetic effects of human gene ITPA and bacterial gene Nfi expression in yeast cells. Also we perform different biochemical studies of listed proteins from human, yeast and bacteria. Our work has a practical importance too. In Humans, the ITPA c.94c>a and g.ivs2+21a>c allelic variants are associated with decreased red cell enzyme activity. The ITPA c.94c>a sequence variant leads to accumulation of ITP and is associated with an increased risk of adverse drug reactions in patients treated with the thiopurine drug azathioprine. The second direction of our work lies in field of mutagenesis too and connected to developing of a test-system for genetic toxicology. The main aim of genetic toxicology is discovering of genetically active environmental agents and endogenous factors as well as estimating the range of their activity. Test systems for genetic toxicology were developed for model organisms from different taxons. An effective testsystem should meet the following requirements: high throughput, high sensitivity to different mutagens, and capability to determinate the different type of damage. Previously we proposed a new test-system in yeast Saccharomyces cerevisiae, which is named the α-test (Kochenova at al., 2007). The α-test is the only test that allows us to distinguish between different types of mutational changes (point mutations, recombination, chromosome or chromosome arm loss) and temporary changes in genetic material. In our work, we have studied resolution and potentials of the alpha-test using mutants defective in repair and replication pathways and different exogenous mutagenic factors.

6 Abstracts of selected publications 1. Stepchenkova E.I., Kozmin S.G., Alenin V.V., Pavlov Y.I. Genome-wide screening for genes whose deletions confer sensitivity to mutagenic purine base analogs in yeast // BMC Genetics :31. Abstract Background N-hydroxylated base analogs, such as 6-hydroxylaminopurine (HAP) and 2-amino-6- hydroxylaminopurine (AHA), are strong mutagens in various organisms due to their ambiguous base-pairing properties. The systems protecting cells from HAP and related noncanonical purines in Escherichia coli include specialized deoxyribonucleoside triphosphatase RdgB, DNA repair endonuclease V, and a molybdenum cofactordependent system. Fewer HAP-detoxification systems have been identified in yeast Saccharomyces cerevisiae and other eukaryotes. Cellular systems protecting from AHA are unknown. In the present study, we performed a genome-wide search for genes whose deletions confer sensitivity to HAP and AHA in yeast. Results We screened the library of yeast deletion mutants for sensitivity to the toxic and mutagenic action of HAP and AHA. We identified novel genes involved in the genetic control of base analogs sensitivity, including genes controlling purine metabolism, cytoskeleton organization, and amino acid metabolism. Conclusion We developed a method for screening the yeast deletion library for sensitivity to the mutagenic and toxic action of base analogs and identified 16 novel genes controlling pathways of protection from HAP. Three of them also protect from AHA. 2. Stepchenkova E. I., Kozmin S. G., Alenin V. V., Pavlov Y. I. Genetic control of metabolism of mutagenic base analogs 6-hydroxylaminopurime and 2-amino-6- hydroxylaminopurime in yeast Saccharomyces cerevisiae // Genetika. V In press Abstract We have studied the influence of mutations in genes of the purine salvage pathway on sensitivity to mutagenic and toxic effect of several analogs of DNA precursors 6-hydroxyaminopuriene (HAP), 2-amino-6-hydroxyaminopuriene (AHAP) and hypoxanthine. For mutagenic activation of HAP and AHAP activity of specific phosphorribosiltransferase, encoded by the APT1 gene is absolutely required. Mutations in the ADE12 and ADE13 genes blocking last step of purine biosynthesis (AMP formation) lead to hypersensitivity to HAP, AHAP and hypoxanthine in yeast.

7 3. Kochenova O.V., Borhsenius A.S., Stepchenkova E.I., Inge-Vechtomov S.G. Genetic control of matting type in yeast Saccharomyces cerevisiae as a base for development of test-system (alpha-test) to estimate genetic activity of endogenous and exogenous agents. // Complied works of the Biological Institute. 54 «Fundamental bases of innovative biological projects in «Naukograd», Russian Abstract Previously we proposed a new test-system in yeast Saccharomyces cerevisiae, which was named the α-test. This system is based on illegitimate hybridization of heterothallic yeast strains of the same mating type α. The α-test is the only test that allows us to distinguish between different types of mutational changes (point mutations, recombination, chromosome or chromosome arm loss) and temporary changes in genetic material simultaneously by genetic analysis of illegitimate hybrids and cytoductants. The main goal of the project is development and calibration of a sensitive test-system for genetic toxicology. We treated tester-strains with several well known and new genotoxyc agents. We have achieved that UV, EMS and oxog induce the illegitimate mating of yeast (αxα) with high frequency. At the same time those mutagens change the ratio of different classes of illegitimate hybrids and cytoductants. While HAP doesn t influence the frequency of illegitimate hybridization and cytoduction. The described test has a good potential to be used in screens for mutagenic and genotoxic agents in environment.