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Chapter 9 .Metabolism of nucleotide Section 1. Metabolism of nucleotide 1.the synthesis of purine nucleotide : de novo synthesis salvage pathway ※ de novo synthesis: The pathway which uses phosphoribose (R-5-P),amino acid ,one carbon unit and CO2 as materials and goes through a series of enzyme-catalized reactions to synthesize purine nucleotide is called de novo synthesis . ※ salvage pathway: The pathway which uses free(ready-made) purine or purine nucleoside as materials and goes through simple reactions to synthesize purine nucleotide is called salvage pathway . (1) the course of de novo synthesis: 1 IMP synthesis: ○ ※ Characteristic: Purine nucleotide is synthesized stepwise on the phosphorobose (R-5-P) molecule rather than synthesizing purine base along fristly and then binding to phosphoribose. ※ From “sweet” to “bitter” (2) salvage pathway: APRT Adeline (reade-made) + PRPP AMP+Ppi HGPRT Inosine (reade-made) + PRPP I MP+Ppi HGPRT Guanine (reade-made) + PRPP GMP+Ppi Lack of HGPRT induce Lesch-Nyhan Syndrome. (3) the sythesis of deoxyribonucleotide: NMP+ATP NDP+ADP Ribonucleotide reductase NDP dNDP (4) antimetabolism of purine nucleotide: 6MP(6-mercaptopurine) is similar to inosine azaserine is similar to Gln aminopterin , MTX is similar to FH 2.Catabolism of purine nucleotide : AMP Isosine Xanthosine Uric acid ※ GMP Adenine Excessive uric acid induce Gout. We use allopurinol to treat the Gout in clinic because allopurinol is similar to inosine. Section 2. Metabolism of pyrimidine nucleotide 1.the synthesis of pyrimidine nucleotide : de novo synthesis salvage pathway (1) de novo synthesis: ※ CPSⅡ(it locate in cytoplasm) CPSⅠ(it locate in Mit) (urea cycle) CO2+Gln carbamoyl phosphate characteristic: ※ Pyrimidine nucleotide is synthesized stepwise purine base along fristly and then binded to phosphoribose rather than synthesizing pyrimidine stepwise on the phosphorobose (R-5-P). ※ From “bitter” to “sweet” (2)salvage pathway: pyrimidine+PRPP pyrimidine nucleotide PPi 2.antimetabolism: 5-Fu(fluorouracil) is similar to 3.catabolism: Cytosine Thymine. Thymine Uracil CO2+NH3 H2N-CH2-CH2-COOH β- Ala H2N-CH2-CH-COOH CH3 β-aminoisobutyric acid ※ Department 3. Transfer of genetic information Gene : Gene is DNA functional fragment encoding biological active products which mainly are protein , various RNA. The central dogma: transcription replication DNA translation RNA protein reverse transcription RNA Chapter 11. Replication (the biosynthesis of DNA) Replication is a process in which genetic information is transmitted from parental DNA to daughter DNA molecules. Section 1. Semi-conservation replication 1.concept : The original double-stranded DNA opens up and both strands serve as template for the synthesis of new DNA. The products of the reaction are two daughter double –stranded DNA molecules each of which has one original template strand and one strand of newly synthesized DNA. 2.experimental evidence : F0 F0 F1 F2 3.the signification of semi-conservation: fidelity F1 F2 Centrifugation Section 2. Enzymes in DNA replication 1. the reaction of replication: (dNMP)n+ dNTP (dNMP)n+1 + PPi or dAMP-dTMP –dGMP + dCTP dAMP-dTMP -dGMP- dCMP +PPi 2. DNA polymerase (DNA-dependent DNA polymerase, DDDP):※ (1) reactive properties: a. dNTP as substrate b. single strand DNA as template c. free 3’-OH as primer d. synthetic direction of new strand is 5’ 3’ (2)In prokaryote cells: polymerase a. Polymerase Ⅰ: single peptide chain, the function is proofreading and repairing. 1 DNA polymerase (5’→ 3’) activity: fill the gap caused by exonuclease Polymerase ○ activity recognizes the next deoxynucleotide on the DNA template and then adds a complementary dNTP to the 3’-OH of the primer ,creating a 3’,5’phosphodiester bond and releasing PPi(pyrophosphate). 2 3’→5’exonuclease activity:it can remove mismatched deoxynucleotide ○ starting from the 3’end of a chain 3 5 ’ →3’exonuclease activity:it can hydrolyze nucleic acid primer ○ starting from the 5’end of a chain Proteinase hydrolyze polymerase Ⅰ into two fragment, larger one and smaller one .The larger one has the activity of DNA polymerase activity and 3’→5’exonuclease activity, also called Klenow fragment ,which can be use as “tool enzyme” in molecular study. b. polymerase Ⅱ:only has its activity without DNA-pol Ⅰand Ⅲ c. polymerase Ⅲ:It is the most effective polymerase in synthesis of new strand DNA. It consist of 10 subunit .αεθ subunit consist of core enzyme 1 DNA polymerase (5’→ 3’) activity: it has ○ 2 3’→5’exonuclease activity ○ (3). In eukaryote cells: DNA polymerase α β γ δ ε DNA pol α: extend the lagging strand DNA pol Ⅲ DNA pol δ: extend the leading strand locate in nucleus DNA pol ε: is similar to DNA pol Ⅰ DNA pol β: only has its activity without other enzyme DNA pol γ: exist in Mit locate in Mit (4). Exonuclease and fidelity: 1 exonuclease: ○ Polymerase Ⅰ degrade DNA in two terminus, so it has the activity of 3’-exonuclease and 5’-exonuclease which can remove the mismatch deoxynucleotide and primer RNA. DNA pol Ⅰalso has the activity of polymerase, so it can fill the gap caused by exonuclease. 2 Proofreading: The course that polymerase Ⅰremove the mismatch deoxynucleotide ○ and then add the correct deoxynucleotide to the gap are called proofreading 3 ○ fidelity and base select: Dependence of DNA polymerase on template ,make the parent DNA and daughter DNA matched precisely, confirm correct transfer of genetic information. Fidelity of DNA is dependent on 3 mechanisms: a. absolute base paired regulation b. base selective function of polymerase on DNA elongation c. proofreading function when mistake happened in DNA replication 3.helicase and topoisomerase: (1)helicase: Unwinding of double helix DNA is required before replication, because the template must paired with its complement dNTP. Helicase can use the ATP to unwind double helix DNA forming a replication fork. Three mainly kinds of helicase: a.DnaA :recognize the origin of replication b.DnaB :true helicase, open double helix helicase c.DnaC :cooperate with DnaB (2)topoisomerase: During unwinding double helix DNA, the downstream double helix DNA over warpped. If DNA over wraped ,called positive-superhelix ,and if wrapped not enough ,called negative-superhelix. There are two kinds of topoisomerase: It can break phosphodiester bond of just one strand of the duplex and then rejoin it, thus unwind supercoiled (positive-superhelix) DNA. DNA topoisomerase break the double helix , unwind supercoiled DNA(when ATP presence). positive-superhelix negative-superhelix positive-superhelix (topoisomerase Ⅰ) (topoisomerase Ⅱ) (3)SSB(single-stranded DNA binding protein):When DNA was unwinded into two single strand ,if paired base existed, it intend to form double-stranded DNA again.SSB binds to single strand DNA and keep the single state of DNA in replication. + DNA SSB 4.primase and primosome (1) primase:※ The replication is initiated by the formation of a short RNA(approximately five nucleotides long) which served as the primer for DNA polymerize. The synthesis of this short fragment RNA was catalized by primase , using DNA as template , so primase is DNA dependent DNA polymerase (DDDP). Primer provide the free3ˊ-OH for the polymerize of the first deoxyribonucleotide . (2) primosome Complex of Dna A. B. C , replicative factors , binding to DNA template , called primosome . Why primer is RNA instead of DNA ? DNA polymerase can not catalyze the polymerize of two free deoxyribonucleotide ,but RNA polymerase can. 5. DNA ligase: During replication , the synthesis of new strand may be discontinuous , the DNA ligase can link two adjacent terminus by 3’-5’phosphodiester bond . It is the “tool enzyme” in genetic engineering. 5ˊp 3ˊ-OH 5ˊp 3ˊ-OH ATP DNA ligase ADP O ‖ 5ˊp O — P — O 3ˊ-OH | O- Section 3 .The course of biosynthesis of DNA Replication is a continuous process , to describe it clearly , we separate it into three stage : initiation , longation , termination artificially . 1. initiation of biosynthesis First step : helicase unwind of double helix DNA forming a replication fork. It include: DnaA recognize the origin of replication DnaB : open double helix DnaC :cooperate with DnaB Second step: topoisomerase unwind supercoiled (positive-superhelix) DNA Third step: SSB binds to single strand DNA and keep the single state of DNA in replication. The forth step: primase synthesize primer RNA (provide the free3ˊ-OH) concept: 1) bi-directional replication in prokaryote cell , initiation started in single ori C site , extending in two direction , called bi-directional replication . origin C 2) replication fork the double helix opens up , and both single strands served as template for synthesis of new DNA , forms “Y” shape , called replication fork . 3)replication bubble (replication eye ) : the region of replicating DNA associated with the single origin is called replication bubble . each replication bubble consists of two replication forks moving in two opposite direction . DNA synthesis proceeds until the replication bubble merge together . 4) replicon in eukaryote cell , many origins form many replication bubbles at the same time , the DNA replicated under the control of single origin is called a replicon . replicon (20-80 units) replication unit 5)replication unit : the replication origins are activated in clusters , called replication unit , consisting of 20-80 origins . 6) primosome: original region together with helicase , Dna C protein , primerase , the complex is called primosome . 7) leading strand and lagging strand※ Double strand DNA is antiparallel , one strand runs 5ˊ-3ˊ and the complementary strand runs 3ˊ-5ˊ. As the original double-stranded DNA opens at a replication fork , new strand is made against each template strand .Therefore , on the template strand with 3ˊ-5ˊ orientation , new DNA is made in a continuous piece in the correct 5ˊ-3ˊ direction , called leading strand . On the other template strand (that has 5 ˊ -3 ˊ orientation) , DNA polymerase synthesizes short pieces of new DNA in 5ˊ-3ˊ direction , and the joins these pieces together by ligase . The new strand which is made by this discontinuous method is called lagging strand . 5’ helicase 5’ 3’ 5’ 3’ lagging strand okazaki fragment SSB Leading strand 5’ 3’ 2.elongation the chemical essence, is the formation of phosphodiester bond . the enzyme catalyze this reaction , is DNA-pol Ⅲ in prokaryote cell , and DNA-polαδ in eukaryote cell . (1) DNA-pol δ : catalyze the formation of long new strand DNA or catalyze the continuous replication in leading strand . DNA-polα : catalyze the formation of short new strand within hundreds of nucleotides or catalyze the discontinuous replication in lagging strand . (2) DNA-pol Ⅲ catalyze synthesis of both strand , which is carried out by different subunit . (3) okazaki fragment※ In the process of replication , the synthesis of leading strand is continuous , in lagging strand , DNA polymerase synthesize short pieces of new DNA in 5ˊ-3ˊ direction , and then joins them together . The discontinuous fragments in lagging strand are called okazaki fragment . (4) rolling circle replication Special form of DNA replication in simple life-form (plasmid) , or DNA outside of chromosome. At first , a nick show in one strand , 5ˊ- terminal stretch out . single strand serve as template , the synthesis of new strand is discontinuous .the other one has no nick , can replicating when circling , the synthesis of new strand is continuous . No primer is needed . 3’ 3’OH 5’ 3’ 3. termination of replication 5’ when replication finished , the 5ˊ- terminal of okzaki fragment is RNA instead of DNA ,so this primer must be cut out , and DNA should fill it , then join these fragment together at last . Mmany kinds of enzymes involved in this process : 1) RNase hydrolyze RNA primer, gap show between fragments 2) DNA-pol Ⅰ catalyze the DNA synthesis in gap , until it was filled up , not DNA-pol Ⅲ DNA-pol Ⅰuses its 5ˊ-3 ˊexonuclease activity to remove the RNA primer and then fills the gap with new DNA 3) DNA ligase when the new synthesis DNA reachs the next fragment , it is stop. The two free terminals form a nick , DNA ligase joins them using ATP . 3’ 5’ 5’ 3’ RNase or pol Ⅰ(5ˊ-3 ˊexonuclease activity) 3’ 5’ 5’ 3’ 5’ 3’ pol Ⅰ( polymerase activity) 3’ 5’ 5’ 5’ 3’ DNA ligase 3’ 5’ 5’ 4. telomere and telomerase in eukaryite cell (1) telomere Telomere is a structure at the terminus s of linear DNA molecule in eukaryote cell . It is important for chromosome to maintain its stability and integrity , also called “bio-clock”. Telomere is the G. C – rich repeated nucleotide sequence . (2) telomerase ※ Telomerase is the complex of RNA and protein . The protein is RNA dependent DNA polymerase , it is a kind of reverse transcriptase . The RNA is template. So telomerase can add DNA repeats(six deoxynucleotide) to the end of eukaryotic chromosome . Section 4 Impair and repair of DNA Mutation ( DNA damage ) : the change of structure , replication and phenotype in one or more deoxyribonucleotide , is called mutation . that is , the change of genetic materials cause the change of genetic information . 1 significance of mutation (1) foundation of evolution & differentiation (2) no phenotype change mutation (3) lethal mutation (4) cause of genetic disease 2 factors for mutation (1)spontaneous mutation rate : 10 –9 (2) induced mutation 1) physical factor a. UV ( ultra violet ) : cause convalent combination of adjacent pyrimidine in DNA chain , form pyrimidine dimer . eg: thymine dimer b. radiation & radiator : X-ray , isotope 2) chemical factor a. base analog : 5-BU b. hydroxylamine c. nitrite d. alkylating agent eg : nitromins 3 type of mutation ※ (1) mismatch (point mutation) , transition and transversion (2) deletion , insertion and frame-shift mutation (3) rearrangement : large fragment exchange in DNA molecule transposition & exchange recombination 4 DNA damage repairing (1) light repairing (2) excision repairing: most important repairing mechanism in cell . mutation indonuclease 5’ pol Ⅰ 5’ 3’ pol Ⅰ ligase 5 3’ (3) recombination repairing 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ (4) SOS repairing Section 5 .Reverse transcription & reverse transcriptase ※Reverse transcription is a process in which genetic information is transmitted from RNA to DNA . Reverse transcriptase is a kind of RDDP . reaction process : reverse transcriptase RNase integration RNA template duplex double strand DNA integration : when virus infects the living cell , it can join host genome by recombination in some cases , and replicate and express with host cell . integration is a important way for virus to lead cancer , which can not integrate into host without being turned to double helix by reverse transcription retrovirus : RSV HIV . Chapter 12. Transcription (biosynthesis of RNA) Concept: ※ Transcription is a process in which genetic information is transmitted from selected DNA to RNA . ※ The mode of transcription is asymmetric transcription. It has two meanings: First, in the double strand DNA, one strand is copied, but the other strand is not. Second, the strand being copied is not always in the same strand, or sometimes one strand is copied, sometimes the other. ※ The single strand DNA being copied is called template strand or Watson strand. The other strand that is not copied is called coding strand or Crick strand. It has the same sequence as the transcriptive product RNA, except that the RNA contains U instead of T. Compare of transcription with replication: 1. Sameness: Template: DNA Enzyme: polymerase Synthesis direction: 5’ → 3’ 2. Difference: Replication Transcription Mode Semi-conservation replication Asymmetric transcription Template Both strands of DNA Single strand of DNA Substrate dNTP NTP Base pairing A-T C-G A-U A-T C-G Enzyme DDDP or DNA polymerse DDRP or RNA polymerase Primer Need Don’t need Produce DNA RNA Section 1. Template and enzymes. 1. Template : The template of transcription is the single strand DNA template strand RNA coding strand DNA structural gene coding strand template strand 2. Enzyme: transcriptase ( RNA polymerase) or DDRP(DNA directed RNA polymerase) (1)In prokaryote cells: RNA pol from prokaryotes is a large multisubunit enzyme , consisting of five subunits ,which are twoαsubunits, oneβ,one β’and oneσsubunit. The subunit of RNA pol Function α Decide which gene to be transcripted β catalyzed the polymerization of RNA β’ Bind DNA template and then unwind the DNA locally to expose a single-strand template σ Recognize the promoter site concept :The complete RNA pol (include two α-subunits, oneβ,one β’and oneσ -subunit) is called the holoenzyme(α2ββ’σ). RNA pol without the σ-subunit is called core enzyme(α2ββ’). ※ Although core enzyme has catalytic function, it cannot initiate transcription specifically at promoter sites; only the complete holoenzyme can do this. Clinic significance: rifampicin can combine with β’-subunit and inhibit transcription. (2). In eukaryote cells: RNA polymerase ⅠⅡ Ⅲ ※ RNA pol RNA pol Ⅰ The product 45S-rRNA RNA pol Ⅱ hnRNA RNA pol Ⅲ 5S-rRNA,tRNA ,snRNA 3. The recognizing and binding of template and RNA pol ※promoter is the DNA consensus sequence which RNA pol recognized and binded. Two important prompter consensus sequence are found in prokaryote , one located about 10 nucleotides (-10 sequence)upstream of where transcription will begin and one located about 35 nucleotides up stream(-35 sequence). The consensus sequence TATAAT located in –10 sequence is also called Pribnow box. ※ Pribnow box DNA -35 -10 5’ TTGACA TATAAT 3’ promoter promoter Transcriptional start site Section 2.The course of transcription The RNA produced by transcription are primary transcripts which have activities through processing. 1. Initiation (1) In prokaryote cells: ※ RNA polymerase holoenzyme (α2ββ’σ) initiates transcription. This involves recognition of the –10 sequence (Pribnow box) and –35 sequence, and then unwinds the double strand DNA to expose a single strand DNA template that can be copied. Transcription initiation complex=RNA pol(α2ββ’σ)-DNA-pppGpN-OH3’ (2) In eukaryote cells: The initiation of RNA synthesis by polymerase is directed by the presence of promoter site on the 5’ side of the transcriptional start site. The promoter consensus sequence is TATA, also called Hogness box or TATA box. There are other promoters upstream the transcriptive start site, such as CAAT box and GC box , these boxes belong to cis-acting element(it is DNA sequence). But the proteins recognizing and binding the cis-acting element are necessary for the initiation .The proteins belong to trans-acting factor. The trans-acting factor which can bind RNA pol directly or indirectly are called TF(transcriptional factor). 2. Elongation Following initiation ,theσ-subunit dissociates from RNA pol to leave the core enzyme (α2ββ’) that continues RNA synthesis in a 5’ 3’ direction using NTP as precursors. The RNA pol ,DNA template and RNA transcript form a transcription bubble or transcription complex. 3. Termination In prokaryote cells: (1). rho(ρ) factor dependent termination※ It allow recognition of the termination site and stop transcription (2).non-ρ factor dependent termination: Transcription continues until a termination signal is reached. The simplest termination signal is a GC-rich region that a palindrome, followed by an AT –rich sequence. The RNA made from the DNA palindrome is self-complementary and so base-pairs internally to form a hairpin structure followed by a few U residues. UU hairpin 5’ GCCGCCAG C CGGCGGUC 3’ HO-UUUUA GG In eukaryote cells: Section 3. Post-transcriptional modification (in eukaryote cell) 1. mRNA : include splicing , capping and polyadenylation ※ hnRNA mRNA RNA pol Ⅱ catalyze hnRNA synthesis (1) RNA splicing: Concept: # Most structural genes in prokaryote are discontinuous, also called splite gene. # The coding sections of the gene(called exons ) are interrupted by noncoding sections of DNA(called introns) . # RNA splicing is the precise cutting out of intron sequences and joining the ends of neighboring exons to produce a functional mRNA molecule. # In the course of RNA splicing, introns becomes branched at a point to form a lariat RNA. # RNA splicing requires the involvement of small nuclear RNA(snRNA). snRNA that has catalytic activity is also called ribozyme(an RNA enzyme). (2) Capping of mRNA Immediately after synthesis , the 5’ end of the primary transcript is modified by the addition of a methylated guanine cap. (3) polyadenylation Next, the 3’ end of the primary RNA is added a poly(A) tail (about 100-200 A residues). The endonuclease cleaves the RNA transcript at a site of a AAUAA sequence that is called a polyadenylation signal. Poly(A) polymerase then adds 100-200 A residues to the new 3’ end using ATP as precursor. 2. rRNA : 45S-rRNA 5S-rRNA rRNA RNA pol Ⅰ catalyze 45S-rRNA synthesis RNA pol Ⅲ catalyze 5S-rRNA synthesis Most eukaryote have more than 100 copies of these genes that encode rRNA. The genes for 18S , 5.8S and 28S-rRNA are typically clustered together and tendemly repeated which are interrupted by untranscribed spacer DNA. The 45S-rRNA is cleaved to release the 18S , 5.8S and 28S-rRNA molecules. Processing may involve snRNA. 18S-rRNA + protein small subunit of ribosome 5S-rRNA 28S-rRNA + protein large subunit of ribosome 5.8S-rRNA ribosome is the synthesizing factory of protein. 3. tRNA: RNA pol Ⅲ catalyze tRNA synthesis Four different types of RNA processing steps # the 5’ extra sequence is cleaved off by ribonuclease P. # splicing introns that are always located in the anticodon loop # add CCA-OH to the 3’end of Trna # about 10% of the base in tRNA are modified, for example methylation (A→ mA), reduction(U → DHU), conversion of uridines to pseudo-uridine and deamination(A → I).※