The Effect Of Methylmethanesulfonate On DNA And Telomerase Activity Of Saccharomyces Cerevisiae

IntroductionTelomerase is a ribonucleoprotein reverse transcriptase that synthesizes telo-meric DNA sequences using its RNA as template. It can remedy the loss of te-lomere after cellular mitosis, maintain telomere length and stabilize chromosome. Telomerase activity was found to be absent in most normal human somatic cells and S. cerevisiae but present in over 90% of cancer cells. Telomerase is closely related with cell growth, proliferation , senescence and carcinogenesis.Recent reports showed that telomerase activity in S. cerevisiae cells could be significantly up - regulated after exposure to some DNA - damaging agents, which may be related with DNA - damage repair. MMS, a kind of DNA - damaging agent, predominantly methylates ring nitrogens of pyrine, leading to DNA double - strand breaks, point mutation and so on. Telomere of yeast is rich in G and A that is ideal target of MMS. Therefore MMS has potential function of damaging yeast telomere. Our aim was to investigate the effect of carcinogen MMS on DNA and telomerase activity of S. cerevisiae S288C and provide some scientific foundation for the relation between telomerase and cancer.Material and Method1. Material:1.1 Strains; Saccharomyces cerevisiae S288C was provided by Microbiology Research Institute of China Science Academy , regular culture.1.2 Reagents and equipment; MMS, DTT and PMSF were purchased fromHua - Mei company. PCR kit, from Takara company in Japan. All other chemicals were of reagent grade.THZ -95set homoiothermyshake box, Eppendorf microcentrifugal machine, Sorvall centrifugal machine ( Germany ) , HRSP0520 PCR instrument ( Hybaid, USA ) , super clean bench, DY - 1 electrophoresis apparatus, etc.2. Method:2. 1 Culture of S. cerevisiae S288C and determination of cell number.2. 2 Treatment with various MMS concentration of MMS and for different time.2.3 Preparation of yeast DNA, 0. 5% agarosegel electrophoresis, EB staining for observation.2.4 Preparation of yeast telomerase, the activity of telomerase was examined by the method of TRAP, polyacrylamide gel electrophoresis and silver staining.2. 5 Statistic treatment with SPSS soft.ResultsAfter treatment with MMS at various (0. 01 Immol/L) concentration for 72h, we examined S. cerevisiae S288C cells for DNA - damage situation and changes in telomerase activity. The result shows that MMS can induce DNA -damage of yeast cells and the situation of DNA - damage aggravated with increase of MMS concentration. 0.5% agarose gel electrophoresis and EB staining demonstrated that the " tail" of DNA strand was the longest when being treated with 1 mmol/LMMS, which illustrate the severest of DNA - damage. Meanwhile MMS significantly enhanced telomerase activity of treated cells even at relatively low doses (0. Olmmol/L). The maximum telomerase up - regulation was obtained at a concentration of 0. 4mmol/L. A time course experiment of DNA -damage and telomerase activity in S. cerevisiae cells treated with 0.4mmol/L MMS revealed aggravation of DNA - damage situation with time prolongation and levels as early as 12h after treatment beginning and increased up to approximately 1. 5 - fold the control level at 72h. After 96h and 120h , telomerase activitydecreased gradually. When MMS was added directly to S. cerevisiae S288C cell extracts immediately before primer addition, telomerase activity remained unchanged. Thus, a direct influence of MMS on TRAP assay does not seem to be involved in telomerase activation observed after drug treatment.DiscussionThe ends of linear chromosomes are protected by telomeres that consist of double - stranded repetitive sequences complexed with specific telomere - binding proteins. In the yeastSaccharomyces cerevisiae, chromosomes end in ³50 base pairs ( bps) of C, ₃A/TGl ₃ DNA. The properties of conventionalDNA pol-ymerases make it impossible for them to replicate the very ends of linear chromosomes. In most eukaryotes, this end replication problem is solved by the enzyme telomerase, a specialized re vers...