A Biological Network Of Saccharomyces Cerevisiae In Response To Oxidative DNA Damage And Its Relationship To Yeast Cell Chronological Aging

Reactive oxygen species(ROS) include superoxide radical,hydroxyl radical, H₂O₂ and so on.In normal case,low level of ROS is necessary for cell function, because they take part in many important cellular signal transduction.But ROS can accumulate a lot in some special case such as intracellular redox equilibrium maladjustment or ionizing radiation from environment.The elevated levels of ROS pose a significant threat to cellular integrity in terms of damage to DNA,lipids, proteins and other macromolecules.There are an increasing number of reports describing the importance of oxidative stress in the etiology of cancer, neurodegenerative and cardiovascular disease as well as the aging process.Saccharomyces cerevisiae is the most simple single cell model,it has readily manipulable genetic system.Since many cellular processes such as protein folding and quality control system,membrane trafficking,and cellular stress responses are highly conserved in many fundamental aspects between human and budding yeast, pathophysiological processes of quite a few neurodegenerative disorders including Creutzfeldt-Jacob,Parkinson's disease and cancer have been modeled in yeast and useful insights regarding the fundamental mechanisms have thus been gained in the past decades.On the other hand,budding yeast is one of the three model in the molecular study of aging area,and it has many advantage than others.In this thesis, we roundly studied DNA damage caused by oxidative stress and different repair system of aerobic organisms.The mechanism of impact of oxidative DNA damage and genome stability on cell aging was also discussed.The major results of the thesis are as follows:1.Using different mutants of repair system of DNA oxidative stress damage, the net system of cell preventing oxidative DNA damage and keeping genome stability was analyzed and established in view of genetics.By analyzing and comparing,some important mutants from antioxidant system, DNA damage repair system and DNA damage checkpoint system were selected and obtained.The mutation rates of CAN1 gene in normal cultured case were tested. Results showed that the mutation rates of all mutants were higher than wild type RDKY3615 at different degree,which illuminated these genes played an important role in keeping cellular genome stability even in normal physiological conditions. Sensitivities of all mutants to different concentration of H₂O₂ were also analyzed. Results showed all mutants were more sensitive to H₂O₂ than wild type strain at different level,of which CTT1,SRX11,TSA1 from antioxidant system,APN1APN2 from DNA damage repair system,SGS1 from DNA replication checkpoint system and MEC1,TEL1,RAD53,DUN1 from DNA damage checkpoint system gene deletion mutants were especially sensitive to H₂O₂.Theses results illuminated these genes were important for cell against DNA oxidative stress damage and maintaining genome stability.So by means of experiment,we confirmed genes of antioxidant system, DNA damage repair system and DNA damage checkpoint system were responsible for DNA oxidative stress damage,all the three systems formed a complex net to protect DNA from oxidative damage and maintain genome stability.2.The relation between repair system of DNA oxidative stress damage and cell aging was studied by analyzing and measuring chronological life spans of mutants of repair system of DNA oxidative stress damage.Chronological life spans of mutants of repair system of DNA oxidative stress damage were measured by plating stationary phase cells in individual solid YPD plates combined with number of total cells using ultraviolet spectrophotometer.The results showed chronological life spans of all mutants were shorter than that of wild types at different degree.The chronological life span of sgslΔmutant from DNA replication checkpoint pathway was shortest of all mutants.Chronological life spans of mutants from DNA damage checkpoint system such as mec1Δ,chk1Δ,rad53Δand tel1Δwere also obviously shorter than that of wild types,of which mec1Δ, chk1Δ,rad53Δmutants were even shorter than tel1Δ.The chronological life span of tsa1Δmutant from antioxidant system was also shorter than that of wild types,but was no shorter than mutants from DNA replication checkpoint system and DNA damage checkpoint system.In contrast,chronological life spans of mutants from DNA damage repair system had little difference from that of wild type except rad51Δmutant of homologous recombination repair pathway.The phenomenon that chronological life spans of all mutants from repair system of DNA oxidative stress damage were shorter than that of wild types at different degree was also validated by integrality of cell membrane examined by PI staining and cellular metabolic ability examined by FUN1 staining.In the progress of aging,the ability to form colonies of different mutants declined,but most cells remained integrity and showed senile morphology,which was verified by microscope.In all,we discovered there was obvious difference between cell number measured by plating stationary phase cells in individual solid YPD plates and cell number of keeping shape integrity in the culture medium in the process of yeast cell chronological aging.That is to say,there were quite numbers of yeast cells losing the ability to divide and replicate in fitting conditions after they entered stationary phase although they could keep shape integrity for a long period of time.This phenomenon was especially obvious in mutants from repair system of DNA oxidative stress damage.3.The mechanism of the impact of repair system of DNA oxidative stress damage on the chronological life spans of yeast cell was discussed.The levels of ROS inside all mutants were higher than that of wild type and were accumulated over time.The levels of ROS were almost consistent with the length of the chronological life spans,which indicated the possible relation between the accumulation of ROS and the length of chronological life span.The accumulation of DNA damage was also analyzed in mutants and wild type cells.The results showed: Mutation rates of mutants from DNA damage repair system were extremely high,but the chronological life spans of them were not much shorter than wild type.On the other hand,mutation rates of mutants from DNA damage checkpoint system and DNA replication checkpoint were lower comparatively,but the chronological life spans of them were much shorter than wild type.The reason may be:Large numbers of and stochastic accumulation of mutations inside mutants from DNA damage repair system didn't affect the process of chromosome DNA replication.Mutants from DNA damage checkpoint system and DNA replication checkpoint might suffer large-scale change or recomposition of genome structure caused by invalidation of holistic modulatory mechanism which final affected the process of chromosome DNA replication directly or indirectly and caused other checkpoint for example the cell wall morphology checkpoint inactive.All of these caused yeast cells losing the ability to divide and replicate.The results of sensitivity of cell wall to SDS and lysing enzyme showed cell wall of all mutants was more sensitive to SDS and lysing enzyme than wild type.And the sensitivity of cell wall to SDS and lysing enzyme was consistent with the length of the chronological life spans.This implied the shorten of the chronological life spans of mutants was probably because that deletion of genes from DNA damage checkpoint system impacted cell wall morphology checkpoint pathway which caused the cell wall weak and shortened the chronological life spans finally.4.Monitoring oxidative stress and DNA damage induced by heavy metals in yeast expressing a redox-sensitive green fluorescent protein roGFP1-R12.Reactive oxygen species(ROS) causing DNA oxidative damage comes from two kinds of ways:one is from cellular normal physiological metabolism;the other is from outer environment.Redox-sensitive green fluorescent protein(roGFP) was expressed in Saccharomyces cerevisiae.Recombinant cells were evaluated in monitoring the changes in the redox state of living cells when challenged with toxicologically relevant metal ions NaAsO₂ or Pb(NO₃)₂ by measuring emission intensity at 510 nm with a Hitachi F6500 fluorescence spectrophotometer,roGFP expressed in yeast responded not only to typical membrane-permeant oxidants H₂O₂ and reductants DTT,but also to toxicological metal ion-induced intracellular redox changes in a dose-dependent manner.Moreover,exposure of yeast cells to NaAsO₂ or Pb(NO₃)₂ at concentrations that induced redox changes reported by roGFP caused up to 2～3 fold increases in DNA mutation frequency.This mutagenic effect was largely caused by oxidative stress since blocking the production of hydryl radicals with thiourea significantly reduced the mutation rate as well as delayed the cell death.