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1 제 10 장. 핵산의생합성 : 유전암호의복제 원핵세포의 DNA 복제

2 복제와전사 : 세포내유전정보의전달

3 DNA, RNA, Protein polymerization Biological polymerization = Assembly (+catalysis) Replication Transcription Translation DNA RNA Protein Function? What makes a good drug Antibiotic?? Bacteria People

DNA 의반보전적복제 (Semiconservative Replication) 1950

4 DNA 의반보전적복제 (Semiconservative Replication) 1950 후반 : Meselson 과 Stahl 의실험 density-gradient ultracentrifugation 보존적복제는원래의 DNA 이중나선은그대로두고, 이를토대로새로운 DNA 이중나선을만드는것. 방사성동위원소를이용한실험을통해이중나선이두개의사슬로분리되어분리된사슬에새로운사슬이합성되는방식이라는것을발견. 이중나선중한개의사슬은원래상태그대로의 DNA 사슬이고여기에새로운사슬이합성되어새로운이중나선을만드므로이를반보존적복제라고한다.

5 원핵생물과진핵생물의복제 원핵생물 (prokaryotes) : 복제원정한개, 양방향복제진핵생물 (eukaryotes) : 복제원점다수, 양방향복제 - Eschericia coli: 4.7*2*10 6 bp; ~ 2 mm - Human: 2.9*2*10 9 bp; ~ 2 m

6 Eukaryotic Replication bubbles visualised by EM Direction of fork movement

7 pbr322 vector (Bacteria) prset EmGFP

8 DNA 가닥하나는반불연속적으로합성된다 선도가닥 (leading strand) : 주형 (3 5 ) 이용 5 3 합성지연가닥 (lagging strand) : 주형 (5 3 ) 이용 5 3 합성 ( 가닥을돌려서 )- 오카자키단편 (Okazaki fragment: 1000~ 2000 nts) ligase 봉합 DNA 복제 : X-ray 구조

9 DNA 복제 : 핵산 (Mg 2+)+ 중합효소 (Mg2+ 필요 ) 5 3 합성

10 The action of DNA polymerase I Preformed DNA performs two roles: One as template (red), which carries the message to be copied. One as a primer (purple) for attachment of added nucleotides.

11 Phosphodiester bonds linking mononucleotides into nucleic acids. The phosphodiester bonds are between the 3 carbon and the 5 carbon of the second nucleotide. This gives direction to the nucleic acids!!! One end has a free 5 OH The other end has a free 3 OH The 3,5 - phosphodiester bonds are highlighted with green

responsible for the polymerization of the newly formed

12 원핵세포복제 : 중합효소의종류 DNA-Pol I: repair of DNA, short DNA synthesis (repair or lagging strand) DNA-Pol III: responsible for the polymerization of the newly formed DNA strand DNA-Pol II, IV, and V: proofreading and repair enzymes

13 subtilisin

Proofreading drastically reduces the errors made in replication (proofreading : 3 5 exo) Enzyme Synthetic domain Proofreading domain Error rate - proof. + proof.

15 Proofreading drastically reduces the errors made in replication (proofreading : 3 5 exo) Enzyme Synthetic domain Proofreading domain Error rate - proof. + proof. DNA pol I aa N-terminal x10-7 DNA pol III subunit subunit 7 x x10-9 T4 DNA pol C-terminal N-terminal 5 x Rev. transcrip. none 10-5

16 Processivity ( 진행도, 공정성 ) How long enzyme stays bound to substrate DNA polymerase III is highly processive: stays bou nd to DNA and can replicate entire E. coli chromo some without dissociating Beta subunit serves as a sliding clamp that tethers DNA polymerase III to template

Origin-binding proteins Initiation of DNA replication require s recognition and binding of origin by specific proteins DnaA binds to oric in prokaryot e E.

22 Origin-binding proteins Initiation of DNA replication require s recognition and binding of origin by specific proteins DnaA binds to oric in prokaryot e E. coli Origin recognition complex (OR C) binds to ARS in eukaryote y east T antigen binds to origin in SV4 0 (monkey virus: model replicat ion system for eukaryotes) ARS : Autonomously replicating sequence

23 Initiation of replication at oric (E. coli) DnaA binds and begins to melt double helix Helicase (DnaB) continues to separate strands

24 원핵세포의 DNA 복제 : 여러효소의복합반응 ( 교재 ) DnaB=helicase

25 Replication forks 5 RNA primer Primase Okazaki fragments on lagging strand DNA Pol I DNA Gyrase Clamp loading complex Leading strand 5 3 DNA Pol III Beta unit (clamp) DnaB (helicase)

26 Topoisomerase Problem: unwinding DNA (HELICASE) would cause positive supercoils in circular DNA (in front of rep fork) Solution: Topoisomerase (gyrase) nicks (cuts) DNA to relieve pressure Nicks one or both strands of DNA provides a swivel point or axis of rotation GYRASE topoisomerase II introduce negative supercoils in E coli chromosome when DNA unwinds; chromosome becomes relaxed

27 DNA Gyrase Grab Cut/ hold Ligate Back Tyr~DNA Front A2B2

28 Eukaryotic DNA synthesis Similarities: DNA synthesis is very similar in prokaryotes and eukaryotes Many of the same enzyme activities are present Helicases Primases Polymerases Ligases Topoisomerases (gyrase) Differences: Eukaryotic genomes are much bigger Replication in eukaryotes occurs during a small portion of the cell cycle (S phase) DNA in eukaryotes is bound by histones

29 Eukaryotic Replication Table 9.5 원핵생물과진핵생물의 DNA 복제의차이 Prokaryotes Three polymerases I - repair enzyme + IV, V II - unknown III- main polymerizing enzyme Polymerases are also exonucleases One origin of replication Okazaki fragments of residues long No proteins complexed with DNA Eukaryotes Five polymerases - makes primers, begins synthesis - repair enzyme - mitochondrial DNA synthesis - main polymerizing enzyme - polymerizing enzyme, primer removal Not all polymerases are exonucleases S everal origins of replication Okazaki fragments of residues long Histones complexed with DNA

30 Similar functions at bacterial and mammalian replication forks: Function E. coli HeLa/SV40 helicase loading helicase single strand priming sliding clamp clamp loading DnaB DnaC SSB DnaG T antigen T antigen RF-A Pol /primase PCNA RF-C catalysis Pol III core Pol holoenzyme dimerization RNA removal ligation t??? Pol I (fill in) MF 1 exonuclease (RNase H) ligase Ligase 1

Eukaryotic DNA Replication: 1. DNA pol + 2 primase subunits: initiates synthesis of lagging and leading strands. 2. DNA pol : elongates both lagging and leading strands. 3.

31 Eukaryotic DNA Replication: 1. DNA pol + 2 primase subunits: initiates synthesis of lagging and leading strands. 2. DNA pol : elongates both lagging and leading strands. 3. PCNA: (proliferating cell nuclear antigen) acts as processivity factor for leading strand (like clamp); elongation. 4. Replication factors C (clamp loading, ATPase) and A (single strand binding). 5. Topoisomerases I & II: maintains DNA winding. ** In lagging strand synthesis, polymerase first adds a primer and a stretch of about 20 deoxynucleotides. Synthesis is then switched (see below) to polymerase

32 진핵생물복제 : 세포주기와 DNA replication

33 Eukaryotic DNA Polymerases Mass (kd) Native Catalytic core Other subunits Location > , 50, None , 47 Nucleus Nucleus Nucleus Nucleus Mitochondria Associated functions 3' -> 5' exonuclease Primase Properties Processivity Fidelity Replication Repair No Yes Low High Yes No Yes No High High Yes? Yes No High High Yes Yes No No Low Low No Yes Yes No High High Yes No

34 진핵생물복제 : Eukaryotic Replication Figure Initiation of DNA replication cycle

35 Eukaryotic Replication Figure 9.20 Structure of PCNA homotrimer PCNA trimer 구조 (B DNA 결합 ( 환형 ))

36 Sealing DNA : Ligase DNA ligase 5 3 -OH O O-P-O - - O O -P-O -(NAD or ATP) - - O 5 T4 phage ligase uses ATP; E. coil ligase uses NAD. 3 3 O O Ligase + AMP P Phosphodiester bond 3 O O Ligase seals nicks in DNA

37 Eukaryotic Replication :summary Figure 9.21 Basics of a eukaryotic replication fork

38 Repetitive sequence End of the chromosome Shortens with each cycle of replication (40~200 bp) What is a telomere? T n A m G o - minisatellite repeat (telomerase), most eukaryotes Telomere Centromere Telomere Telomere Centromere Metaphase Chromosome

39 Telomeres shorten due to lagging strand synthesis

40 Telomeres are lengthened by telomerase : 암세포에만존재

41 Telomerase (human-dimer)

42 Campbell Chap10 Part B: DNA Mutation AND Repair

43 Oxidative stress or Environmental insults Cell cycle arrest and DNA repair DNA damage repaired Cell cycle progression DNA 손상복구 정상세포 DNA 복구불능 세포자살 / 암 Normal cells Excessive DNA damage Genomic instability Enhanced carcinogenesis Apoptosis 세포자살 Clonal selection of apoptosis resistant cells 암세포화 외부자극 (stress) 에대한생체의대응방법. 43

44 Mechanisms : Spontaneous Mutation Tautomeric shifts-rare isomers of each base pair incorrectly in the DNA helix. Polymerase errors-misincorporation of bases during DNA replication. Depurination-loss of purines from the DNA helix Deamination-loss of the amine group from cytosine (yielding uracil), and from 5-methylcytosine (yielding thym ine). Transposable elements-insertions of mobile DNA natural mutagens

45 Tautomeric forms of bases 호변이성체 : 염기가자연상태에서두가지구조로존재할수있다 : 염기의 keto 또는 enol forms - G & T : keto or enol form - A & C : amino or imino form

46 Mutation; a two-step process

48 Mutagens PHYSICAL CHEMICAL BIOLOGICAL USE Research Disease Warfare 48

49 Physical Mutagens Radiation Non-ionising Ultraviolet (UV), 에너지가낮아이온화는못시킴. o UV-C ( nm) 는에너지가높아치사를유도 (germicidal) o UV-B ( nm) 는 pyrimidine dimer를형성-돌연변이 Ionising : X-Rays 49

50 UV (260nm) Absorbed by bases of DNA and RNA forms pyrimidine dimers 2 adjacent bases (CC or TT) are covalently joined UV 에의한손상피리미딘 2 량체 ( 사이클로부탄형성 ) 50

51 Ionising radiation X-rays, cosmic rays, gamma rays More powerful than UV Penetrates glass 산소레디칼에의한손상 51

52 Chemical Mutagens 1.Base analogues Resemble DNA bases Faulty pairing properties Example 5-bromouracil incorporated like T faulty pairing with G 52

53 2. Intercalating agents Mutagen inserted between two DNA basepairs, pushing them apart Insertions and deletions during replicatioin due to odd shape Example acridine orange, EtBr 3.Alkylating agents Insertions and deletions through cross-linking DNA strands Example nitrogen mustards 53

54 Biological Mutagens Insertion of a transposon( 이동유전자, jumping genes) within a gene disrupts the reading frame loss of function Jumping genes- transposable elements move to different positions in the chromosome. Transposon carries other genes with it. Transposon 연관잘병 - 혈우병 A( 인자 VIII 유전자 ), 혈우병 B( 인자 IX 유전자 ) - X 연관악성면역결핍증 (SCID) : IL-2 수용기유전자 - 포르피린증 : 혈색소과다로인한포르피린증가 - 직장암에잘걸리는체질 (predisposition) : APC 유전자 - 던쳔근디스트로피 : 근위축증유전자 54

55 DNA 의교정과수선 (Proofing and Repair) 한세대에수차례일어나는 RNA 나단백질의합성과정과는달리 DNA 복제는평균적으로각세포에서한번만일어난다. 이경우복제과정의충실도를높이기위해세포는교정 (proof) 과수선 (repair) 을한다. 그림 9.11 중합효소 I 의 3 5 핵산외부가수분해활성 : 교정및수선역활

56 Proofreading by DNA polymerase III (Prokayote)

57 Repair Processes O Light Dependent Repair -reversal of dimers by photolyase. O Mismatch Repair -following replication, mismatches are identified and the misincorporated base is removed and resynthesized with correct base. O Excision Repair -base(s) is(are) removed by DNA glycosylase, leaving AP site, which is then repaired by general excision pathway. NER(nucleotide excision repair) BER(base excision repair) O Recombination Repair -use of a good strand from sister chromatid to patch across from bad template.

58 DNA 오류쌍수선 (mismatch repair): 본가닥을메칠화, 잘못된가닥 ( 메틸화안된것 ) 을잘라냄

염기추출수선 ( 절단수선, excision repair) Base Excision Repair

general, exonuclease human disease defective in NER

59 염기추출수선 ( 절단수선, excision repair) Base Excision Repair (BER) ; the damaged base is removed Nucleotide Excision Repair (NER); Bulky DNA damage is removed - in E. coli, UvrABC endonuclease (12-13 nt) - in general, exonuclease human disease defective in NER ; Cockayne s syndrome, 코케인증후군 Xeroderma pigmentosum (XP), 색소성건피증

60 NER 결손에의한질병 색소성건피증 Xeroderma pigmentosum (XP) NER 결손에의한 Colon Cancer 의생성

61 Nucleotide excision repair : NER

62 Base excision repair : BER 염기제거 cut 중합 당, 인산제거 ligation

63 Base loss Abasic site -loss of a nucleobase (apurinic or apyrimidinic) Deamination C U

64 Base Excision Repair

65 . Short-patch 와 long-patch BER 에관련된단백질들. (Nature, 411: , 2001)

Chapter 14 유전정보 (Genetic Information) A + G = C + T 일정 ( 샤가프룰 ] - Watson & Crick(1953년 ) : double helix model of DNA( 이중나선구조 ) 제시 1. 유전정보의전달경로 DNA 상의정

Chapter 14 유전정보 (Genetic Information) A + G = C + T 일정 ( 샤가프룰 ] - Watson & Crick(1953년 ) : double helix model of DNA( 이중나선구조 ) 제시 1. 유전정보의전달경로 DNA 상의정보를 RNA로옮겨주는과정 ex) retrovirus 2. DNA Replication ( 유전자복제