生物化學(xué)資料：怪盜基德與DNA最后的決戰(zhàn)

1、怪盜基德與DNA最后的決戰(zhàn),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, please,TOPICS OUTLINE,Topic 1 : Reverse transcription,Reverse transcriptase (RNA-dependent DNA polymerase,DNA-dependent RN

2、A polymerase,In normal eukaryotes,In the host of retroviruses,Telomeres and telomerase,Retrotransposons,During the replication of retroviruses,Existence,Movement 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

3、 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 replic

4、ative transposon which involves an RNA intermediate,Reverse transcriptase,DNA-dependent DNA polymerase,RNase H (H=Hybrid,RNA-dependent DNA polymerase,Function/activity,crystal structure of HIV reverse transcriptase,Process,Take the HIV, a kind of retroviruses, as an example,Reverse transcription,App

5、lication of reverse transcription,RT-PCR (Reverse Transcription-Polymerase Chain Reaction,Primer,Oligo dT,Random hexamer,Gene-specific,Application,Levels of gene expression,RNA viruses,DNA clone,Topic 2 : Organization of DNA,A typical human cell contains 46 chromosomes, whose total DNA is approximat

6、ely 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 DNA,Take E

7、.coli as an example,Closed-loop molecules,4.6Mb in length,Distribute intensively in nucleoid,Plasmid,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 domains,The nucleoid concentrates the DNA in p

8、art 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 DNA,Via folding and compressing step by step to form an advanced structure, eukaryotic genome exists in the nucleus in the fo

9、rm 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 nucleosomes,Histones:As a result of their high content of lysine and arginine,histones are positively charged at p

10、hysicologic 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 approximate

11、ly 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,Solenoid,The 3

12、0 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 levels,Topic 3 : DNA damage and repair,DNA damage (DNA lesion): An alteration to the normal chemical or physical st

13、ructure 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 damage,a. Errors during DNA replication,b. Instability of DNA,c. The pro

14、duction of reactive oxygen species (ROS) during metabolism,In vivo (spontaneous,a. Errors during DNA replication,Mismatches of bases (1/1010,Tautomeric shift of bases,Concentrations of dNTP,Loss or insertion of DNA fragments (slippage,Especially the short repetitive sequences,Result in neurodegenera

15、tion,Huntingtons disease(chorea,Fragile X syndrome,Myotonic dystrophy,b. Instability of DNA,the most important & frequent,Heat / changes of pH,Bases deletion (usually purines,break the,glycosidic linkage,Deamination,Transformation of bases,Deamination is the removal of an amine group from a molecule

16、. Enzymes that catalyse this reaction are called deaminases,c . ROS,Oxidize the bases directly,Factors of DNA damage,In vitro,a . Physical factors,b . Chemical factors,c . Biological factors (viruses,a . Physical factors,Break the double-strand DNA directly,Stimulate free radical reactions,High-ener

17、gy ionizing radiation,Nonionizing radiation (e.g. ultraviolet, UV,Do damage to DNA (260nm,Stimulate the formation of pyrimidine dimer,Lead to cross-linking and rupture in dsDNA,b . Chemical factors,Free radicals,Base analogs,Embedded dyes,Deamination(HNO2,oxidation(.OH) or reduction(.H,keto match to

18、 A,enol match to G,inserted into base pairs,Base modifiers,5-bromo uracil (5-BU,Alkylation,Categories of DNA damage,Damage to the bases or sugars,Mismatch of bases,Rupture of DNA,DNA covalent cross-linking,specific glycosylases,AP site (apurinic-apyrimidinic site/abase site,Transition (轉(zhuǎn)換,Transversi

19、on(顛換,purine pyrimidine,purinepurine or pyrimidinepyrimidine,Single base mutation,Addition or deletion of base,This 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 bases,DNA repair,b . Exci

20、sion repair,c . Recombinational repair,d . Damage bypass,a . Direct repair,a . Direct repair,Pyrimidine dimer (not the main pathway,photoreactivation with photolyase (300-500nm,especially 400nm,Alkylated bases,alkyl-transferase which can transfer the alkyl onto its own peptide,Apurinic sites,DNA pur

21、inic insertase(K,Single-strand breaks,DNA ligase,pyrimidinic insertase,b . Excision repair,Base excision repair,BER,Hydrolysis glycosylase,Recognition and excision AP endonuclease,Gap-filling DNA polymerase,Nick-filling DNA ligase,Mismatch repair,MMR,Discrimination Mut proteins based on the degree o

22、f methylation,Recognition A group of enzymes,Excision Endonuclease,Gap-filling DNA polymerase,Nick-filling DNA ligase,recognize the twists resulting from the damage to DNA,Global genome NER,GGR,Transcription-coupled NER,TCR Recognized by RNA polymerase,The main pathway of pyrimidine dimer repair,Xer

23、oderma pigmentosum,XP,Nucleotide excision repair,NER,c . Recombinational repair,Homologous recombination repair,HR,Nonhomologous end-joining recombination repair,NHEJ,Double-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 mutagenic,RAD51 protein,replication protein A,RPA,Homologous rec