DNA synthesis and repair 2557 Outline DNA replication
- Slides: 64
DNA synthesis and repair กลลประวทธ รชนกร 2557
Outline • DNA replication • DNA polymerase and other proteins in DNA replication • Telomere and telomerase • DNA synthesis using RNA template • Damage to DNA and mutation • DNA repair defect 2
1 A A C T G G T T C C A 2 T T G A C 1 A A C T G G T T C C A T T G A C C A A G G T C C C A A G G T A A C T G G T T C C A 2 T T G A C C C T A A G G T G A Semiconservative replication 7
DNA replication • Unwinding of DNA helix : helicase • Relaxation of supercoiled DNA : DNA gyrase • RNA primer synthesis : primase • DNA synthesis : DNA polymerase III – Leading strand – Lagging strand • Removal of RNA primer & filling gaps : DNA polymerase I • Sealing of DNA chain : DNA ligase 10
Replication begins with the unwinding of the Ori C (origin site) E. coli Tandem array of 13 -mer seq. Binding site for Dna. A protein 5' GATCTNTTNTTTT 3' 3' CTAGANAANAAAA 5' Ori C locus - 245 bp - tandem array of three 13 -mers with nearly identical sequences (AT rich) Dna A binds to Ori C initiate sequence of steps of DNA synthesis 11
HU : histone like protein stimulates initiation 12
Origin of replication Replication forks New strands Parental strands Bidirectional replication of a circular chromosome Replication เรมเกดท origin of replication โดยเกดทง 2 ทศทางในขณะเดยวกน 13
3' 5' 5' A T T G C A T A A C G T OH DNA ligase 3' H 2 O 3 PO ATP AMP +PPi H 2 O 3' 5' 5' A T T G C A T A A HC O O P O O G T 3' 23
DNA polymerase • catalyzes a template-directed synthesis of DNA from d. NTP • requires free 3'-OH group • synthesis in the 5' 3' direction • Uses d. ATP, d. GTP, d. CTP, and d. TTP as substrates 24
DNA polymerase • DNA polymerase in prokaryotes (E. coli) – DNA polymerase III • DNA polymerase in eukaryotes 25
DNA polymerase I • 10 nucleotides / sec / mol-enzyme • Polymerase activity (5’ 3’) • 3’ 5’ exonuclease activity ทำหนาท proofreading • 5’ 3’ exonuclease activity ทำหนาท error-correcting • ใชใน DNA synthesis (ตอ Okazaki fragment), remove primer, filling gaps, DNA repair 26
DNA polymerase I ม activity ของ enzyme 3 ชนด อยในตวเอง 1. 3' 5' exonuclease proof-reading 2. 5' 3' exonuclease error-correcting, remove primer 5' H^ 2 O site 3' HO C T T T A A A A 3' 5' C H^ 2 O site 5' 3' 3. polymerase N 3' C 5' 3' exonuclease small fragment 3' 5' exonuclease C T T T A A A 5' polymerase large fragment ( Klenow fragment ) C 27
DNA polymerase II • Multisubunit enzyme (>4 subunits) • 40 nucleotides/ sec/ mol-enzyme • 3' 5’ exonuclease activity ทำหนาท proofreading DNA repair 28
DNA polymerase III holoenzyme • multisubunit enzyme • 1000 nucleotides / sec / mol-enzyme • highly possessive เนองจาก β subunit ชวยยด core enzyme ใหตดกบ DNA template ทำให enzyme ไมหลดจาก DNA template งายๆ ทำใหมการ form phosphodiester bond จำนวนหลายแสน bond กอนท enzyme จะถกปลอยออกจาก template เมอ replicate เสรจ • Replicative chain elongation (leading & lagging strand) 29
DNA polymerase III 3' 5’ exonuclease activity ทำหนาท proofreading activity ชวยปองกนไมใหเกดการ mutation ระหวางทม DNA replication 30
DNA polymerases in eukaryotes (e. g. humans) 1. DNA polymerase α (replication)* – สงเคราะห short primer (RNA or DNA) for Okazaki fragments on a lagging strand 2. DNA polymerase β (repair)* 3. DNA polymerase γ (replication of mitochondrial DNA)* ทง 3 ชนดไมม 3' 5' exonuclease or proofreading activity 4. DNA polymerase δ (replication) อยใน nucleus ม 3' 5' exonuclease or proofreading activity – สงเคราะห leading และ lagging stand (คลาย DNA pol III ใน prokaryote) 5. DNA polymerase (repair, replication – RNA primer removal คลาย DNA pol I ใน prokaryote) หลงจากสงเคราะหจะม methyl groupใหกบเบส modification ของ DNA โดยการเตม เชน C 5 ของ cytosine ดวย SAM 31
RNA dependent DNA polymerase DNA synthesis using RNA template • เกดโดยเอนไซม reverse transcriptase (RNA dependent DNA polymerase) • พบในไวรสบางชนด เชน ไวรสททำใหเกดโรคมะเรง (Rous sarcoma, Avian myeloblastosis, Raucher murine leukemia), ไวรสททำใหเกดโรคเอดส (HIV) 32
การสงเคราะห DNA ทใช template RNA เปน retrovirus RNA reverse transcriptase (RNA depentdent DNA polymerase) RNA DNA สลายสวนทเปน RNase. H RNA ออกไป โดย DNA 33
Area where gap is generated upon replication Parental Leading Parental Lagging New DNA 3' 5' การถอดแบบ DNA ของโครโมโซมทเปนเสนตรงไดเปนสาย คอ สาย leading และ สาย lagging 3' 5' DNA ใหม 2 สาย 36
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Parental strand AGGGTT Short repeats making up telomere AGGGTT 3' Newly synthesized lagging strand 1 Telomerase binds New telomere repeat 2 Telomerase extends 3' end TCCCAA AGGGTT reverse transcriptase AGGGTT UCCCAA TCCCAA RNA template 3 Telomerase released AGGGTT UCCCAA TCCCAA 4 Primer is bound. DNA polymerase completes lagging strand AGGGTT TCCCAA Telomerase AGGGTT DNA polymerase AGGGTT TCCCAA UCCCAA Primer Extended telomere permits completion of lagging strand 39
DNA repair 41
Damage to DNA • Force distortion, bending, breaking • X-rays, γ -rays , ionized radicals • activated oxygen • ultraviolet light - thymine dimer • intercalation, cross-link by foreign compound (some chemicals, drugs) • accident of replication • spontaneous decay of DNA (loss of base) 42
Mutation • base substitution – transition : Pu 1 – transversion : Pu Pu 2, Py 1 Py, Py Py 2 Pu • frameshift mutation – deletion – Insertion ATC GTA TTA ATA … ATC GAT TAA TA … T ATC GTA TTA ATA … ATC GTT AAT A. . 43
A T G C C A T A A T transition A G C T A T C A T deletion (G) A T G C C T (G) T A A T transversion A G C T C G C T A A T insertion 44
Trinucleotide repeat expansion (triplet repeats) • การเกดขนซำ ทพบบอยคอ ๆ กนของ nucleotide 3 ตว CGG, CAG และ CTG • Triplet repeat อาจอยใกลกบ gene หรออยใน gene • มกทำใหเกดโรคทางระบบประสาท • ตวอยางโรคทเกดจาก triplet repeat ไดแก fragile. X symdrome, spinobulbar muscular dystrophy, myotonic dystrophy และ Huntington’s disease 46
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Fragile X syndrome 49
Normal DNA Damaged DNA Endo and exonucleases DNA with damaged region removed excinuclease P OH Repaired DNA polymerase Nick OH P DNA ligase Repaired DNA (normal) Common steps in DNA repair mechanisms. 51
Mismatch repair methylated parental strand CH 3 3' 5' 5' 3' CH 3 unmethylated newlysynthesized strand 3' 5' 5' 3' DNA segment replaced 53
Normal DNA b a s e e x c i s i o n r e p a ir AP site glycosylase n u cl e o t I d e Damage to bases incision endonuclease e x ci s i o n r e p ai r excision endonuclease AP site – Apurinic site – Apyrimidinic site DNA polymerase DNA ligase Repaired (normal) DNA 56
P O deoxyribose N H O P deoxyribose U. V. deoxyribose P deoxyribose O CH 3 H N N H O P N N H O N H H O CH 3 N H การเกด thymine dimer (cyclobutane ring) เมอ DNA ถกแสง UV O CH 3 H N O CH 3 57
thymine dimer 58
Repair of thymine dimer 5' 3' 3' 5' excision of a 12 nt fragment by Uvr ABC excinuclease DNA synthesis by DNA polymerase 1 joined by DNA ligase 59
Direct repair • การซอมแซม DNA โดยไมตองตดเบสหรอนวคลโอไทดออก • พบใน bacteria ไมพบในมนษย • ตวอยางไดแก การซอมแซม cyclobutane pyrimidine dimer โดยเอนไซม photolyase (photoreactivating enzyme) และ visible light ตด dimer โดยไมสลาย phosphodiester bond T^T AA photoreactivating enzyme (photolyase) and visible light TT AA 61
DNA repair defect • ทำใหเกดโรคทางพนธกรรมไดหลายโรค • ตวอยางโรคทเกดจากความผดปกตของ DNA repair – Xeroderma pigmentosum – Chromosomal breakage syndrome • Bloom’s disease • Fanconi anemia • Progeria 62
RECOMBINATION REPAIR (post-replication repair) 5' 3' Replication 3' 5' DNA containing a pyrimidine dimer Damaged parental strand Gapped daughter strand Normal duplex DNA Recombination repair Fill in and ligation Damaged parental strand Repaired daughter strand Normal daughter strand Gapped parental strand DNA containing a pyrimidine dimer Normal duplex DNA 64
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