生物化学资料：怪盗基德与DNA最后的决战

怪盗基德与怪盗基德与DNA最最后的决战后的决战If you are familiar with the content of this part, just pretend to be interested and curious about what I will talk about. I have to say.Dont hurt my fragile heart, pleaseTOPICS OUTLINETOPICS OUTLINETopic 1 : Reverse transcriptionReverse transcriptase(RNA-dependent DNA polymerase)DNA-dependent RNA polymeraseDNA-dependent RNA polymeraseIn normal eukaryotesIn the host of retrovirusesTelomeres and telomeraseRetrotransposons During the replication of retrovirusesExistenceMovement mediated by transposase, an enzyme encoded by the Tn itself, can be direct, in which transposase cuts out and then inserts the Tn at a new site, or replicative, in which the Tn is copied and the copy inserted elsewhere while the original remains in place.Transposon (Tn)A mobile segment of DNA that move in an essentially random manner from one site to another on the same or a different chromosome.The retrotransposon is a kind of replicative transposon which involves an RNA intermediate.Reverse transcriptaseDNA-dependent DNA polymeraseRNase H (H=Hybrid)RNA-dependent DNA polymeraseFunction/activitycrystal structure of HIV reverse transcriptase Process Take the HIV, a kind of retroviruses, as an example.Reverse transcriptionApplication of reverse transcriptionRT-PCR (Reverse Transcription-Polymerase Chain Reaction)PrimerOligo dTRandom hexamerGene-specificApplicationLevels of gene expressionRNA virusesDNA cloneTopic 2 : Organization of DNAA typical human cell contains 46 chromosomes, whose total DNA is approximately 2m long! How such a large amount of genetic material can be effectively packaged into a volume size of a cell nucleus so that it can be efficiently replicated, and its genetic information expressed?(spatial structure of DNA) SEM microstructure image of E.coli Organization of prokaryotic DNATake E.coli as an exampleClosed-loop molecules4.6Mb in lengthDistribute intensively in nucleoidPlasmid(d) If the DNA backbone is nicked by a nuclease, the DNA domain opens .(c) The DNA is further condensed by supercoiling . (a) The circular DNA molecule (b) DNA domainsThe nucleoid concentrates the DNA in part of the cell, but it is not separated by a nuclear membrane. The bacterial DNA is packaged in loops to a protein core, attached to the cell wall. Organization of eukaryotic DNAVia folding and compressing step by step to form an advanced structure, eukaryotic genome exists in the nucleus in the form of chromatin. The chromatin is made up of DNA, histones, nonhistones and a small quantity of RNA, serving as the carrier of genetic information and epigenetic information.Histones and nucleosomesHistones:As a result of their high content of lysine and arginine,histones are positively charged at physicologic PH,so that they form ionic bonds with negatively charged DNA.Nucleosomes:Two molecules each of H2A,H2B,H3 and H4 form the octameric core .Around this structural core,a segment of dsDNA is wound nearly twice,causing supercoiling.Neighboring nucleosomes are joined by linker DNA approximately 50 base pairs long.H1 binds to the linker DNA chain betweeen nucleosome beads,facilitating the packing of nucleosomes into more compact structures.Nucleosomes can be packed more tightly to form a nucleofilament.This structure assumes the shape of a coil,referred to as a 30 nm fiber.SolenoidThe 30 nm fiber is organized into loops that are anchored by a nuclear scaffold containing several proteins. Additional levels of organization lead to the final chromosomal structure.Higher levelsTopic 3 : DNA damage and repairDNA damage (DNA lesion): An alteration to the normal chemical or physical structure of DNA . In most cells, an elevated level of DNA damage causes both an increased synthesis of repair enzymes and a delay in the cell cycle to help to ensure that DNA damage is repaired before a cell divides.Factors of DNA damagea. Errors during DNA replicationb. Instability of DNAc. The production of reactive oxygen species (ROS) during metabolismIn vivo (spontaneous)a. Errors during DNA replicationMismatches of bases (1/1010)Tautomeric shift of basesConcentrations of dNTPLoss or insertion of DNA fragments (slippage)Especially the short repetitive sequencesResult in neurodegenerationHuntingtons disease(chorea)Fragile X syndromeMyotonic dystrophyb. Instability of DNA(the most important & frequent)Heat / changes of pHBases deletion (usually purines)break theglycosidic linkageDeaminationTransformation of basesDeamination is the removal of an amine group from a molecule. Enzymes that catalyse this reaction are called deaminases.c . ROSOxidize the bases directlyFactors of DNA damageIn vitroa . Physical factorsb . Chemical factorsc . Biological factors (viruses)a . Physical factorsBreak the double-strand DNA directlyStimulate free radical reactionsHigh-energy ionizing radiationNonionizing radiation(e.g. ultraviolet, UV)Do damage to DNA (260nm)Stimulate the formation of pyrimidine dimerLead to cross-linking and rupture in dsDNAb . Chemical factorsFree radicalsBase analogsEmbedded dyesDeamination(HNO2)oxidation(.OH) or reduction(.H)keto match to Aenol match to Ginserted into base pairsBase modifiers5-bromo uracil (5-BU)AlkylationCategories of DNA damageDamage to the bases or sugarsMismatch of basesRupture of DNADNA covalent cross-linkingspecific glycosylases AP site (apurinic-apyrimidinic site/abase site)Transition (转换转换)Transversion(颠换颠换)purine pyrimidinepurinepurine or pyrimidinepyrimidine Single base mutationAddition or deletion of baseThis can result in a product with a radically different amino acid sequence or a truncated product due to the creation of a termination codon.The power of abnormal basesDNA repairb . Excision repairc . Recombinational repaird . Damage bypassa . Direct repaira . Direct repairPyrimidine dimer(not the main pathway)photoreactivation with photolyase (300-500nm,especially 400nm)Alkylated basesalkyl-transferase which can transfer the alkyl onto its own peptideApurinic sitesDNA purinic insertase(K+)Single-strand breaksDNA ligasepyrimidinic insertase?b . Excision repairBase excision repair,BERHydrolysis glycosylaseRecognition and excision AP endonucleaseGap-filling DNA polymeraseNick-filling DNA ligaseMismatch repair,MMRDiscrimination Mut proteinsbased on the degree of methylationRecognition A group of enzymes Excision EndonucleaseGap-filling DNA polymeraseNick-filling DNA ligaserecognize the twists resulting from the damage to DNAGlobal genome NER,GGRTranscription-coupled NER,TCRRecognized by RNA polymeraseThe main pathway of pyrimidine dimer repairXeroderma pigmentosum,XPNucleotide excision repair,NERc . Recombinational repairHomologous recombination repair,HRNonhomologous end-joining recombination repair,NHEJDouble-strand DNA breaks cannot be corrected by the previously described strategy of excising the damage on one strand and using the remaining strand as a template. Instead, they are repaired with the help of another dsDNA.error prone and mutagenicRAD51 proteinreplication protein A,RPAHomologous recombination repaird . Damage bypassRecombinational bypasswhen damage makes DNA incompetent to be a template of replicationwhen damage appears during DNA replicationSOS repair(error-prone repair)RecombinationalbypassHigher level of DNA damage effectively inhibits DNA replication and triggers a stress response in the cell,involving regulation in the levels of a number of proteins. This is called the SOS resposne.SOS repair(error-prone repair)DNA polymerases with low fidelity and no proofreading activity add nucleotides to the chain randomly.Biodiversity is dependent on the equilibrium between DNA mutation and repair.efficiently repairedmaintaining the normal functioninvalidly repairedchanges of structure and function,even leading to apoptosis or diseases In conclusion