Presentation is loading. Please wait.

Presentation is loading. Please wait.

Molecular Biology Fifth Edition Chapter 20 DNA Replication, Damage, and Repair Lecture PowerPoint to accompany Robert F. Weaver Copyright © The McGraw-Hill.

Similar presentations

Presentation on theme: "Molecular Biology Fifth Edition Chapter 20 DNA Replication, Damage, and Repair Lecture PowerPoint to accompany Robert F. Weaver Copyright © The McGraw-Hill."— Presentation transcript:

1 Molecular Biology Fifth Edition Chapter 20 DNA Replication, Damage, and Repair Lecture PowerPoint to accompany Robert F. Weaver Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

2 General Features of DNA Replication Double helical model for DNA includes the concept that 2 strands are complementary Each strand can serve as template for making its own partner –Semiconservative model for DNA replication is correct –Half-discontinuous (short pieces later stitched together) –Requires DNA primers –Usually bidirectional

3 20-3 Three Hypotheses of Replication The three methods of DNA replication considered were: 1.Semiconservative 2.Conservative 3.Dispersive

4 20-4 DNA replicates in a semiconservative manner When parental strands separate –Each strand serves as template –Makes a new, complementary strand Semiconservative Replication

5 20-5 Semidiscontinuous Replication DNA replication in E. coli (and in other organisms) is semidiscontinuous One strand (the leading strand) is replicated continuously in the direction of the movement of the replicating fork The other strand (the lagging strand) is replicated discontinuously as 1-2 kb Okazaki fragments in the opposite direction This allows both strands to be replicated in the 5 3-direction

6 20-6 DNA Replication Models

7 20-7 Priming DNA Synthesis Okazaki fragments in E. coli are initiated with RNA primers nt long Intact primers are difficult to detect in wild-type cells because of enzymes that attack RNAs

8 20-8 Bidirectional Replication The replication structure resembles the Greek letter, DNA replication begins with the creation of a bubble – a small region where parental strands have separated and progeny DNA has been synthesized As the bubble expands, replicating DNA begins to take on the shape

9 20-9 Theta Mode of DNA Replication in E.coli

10 20-10 Replication Fork In DNA replication, the replication forks represent the sites of DNA replication Direction of replication: –Unidirectional – one fork moving away from the other which remains fixed at the origin of replication –Bidirectional – two replicating forks moving in opposite directions away from the origin Origin of replication is the fixed starting point for DNA replication The replicon is the DNA under the control of one origin of replication

11 20-11 Rolling Circle Replication Circular DNAs can replicate by a rolling circle mechanism –One strand of a dsDNA is nicked and the 3-end is extended –This uses the intact DNA strand as a template –The 5-end is displaced Phage X174 replication cycles so that when one round is complete a full-length, single- stranded circle of DNA is released Phage, displaced strand serves as the template for discontinuous, lagging strand synthesis

12 20-12 Phage Rolling Circle Model As the circle rolls right –Leading strand elongates continuously –Lagging strand elongates discontinuously Uses unrolled leading strand as a template RNA primers for Okazaki fragments Progeny dsDNA produced grows to many genomes before one genome worth is clipped off

13 Enzymology of DNA Replication Over 30 different proteins or enzymes cooperate in replicating the E. coli DNA Examine the activities of some of these proteins and their homologs in other organisms –Start with DNA polymerases – the enzymes that make DNA

14 20-14 E. coli DNA Polymerases There are 3 DNA polymerases, the enzymes that make DNA, found in E. coli: –pol I –pol II –pol III E. coli DNA polymerase I was the first polymerase identified It was discovered in 1958 by Arthur Kornberg

15 20-15 DNA Polymerase I DNA polymerase I (pol I) is a versatile enzyme with 3 distinct activities –DNA polymerase –3 5 exonuclease –5 3 exonuclease –Mild proteolytic treatment results in 2 polypeptides Klenow fragment (the large domain) Smaller fragment

16 20-16 Klenow Fragment Contains both: Polymerase and 3 5 exonuclease activity, which serves as proofreading –If pol I added wrong nt, wont base pair properly –Pol I pauses, exonuclease removes mispaired nt –Allows replication to continue –Increases fidelity of replication

17 20-17 Klenow Fragment Structure Wide cleft for binding to DNA between two -helices like a hand –One helix is part of the fingers –Other helix serves as the thumb domain –Between the helices lies a -sheet, palm 3 conserved Asp residues Essential for catalysis Likely coordinate Mg 2+ (metal ions) Polymerase activity is separated from the exonuclease activity

18 exonuclease This activity allows pol I to degrade a strand ahead of advancing polymerase Removes and replaces a strand in one pass Basic functions are: –Primer removal –Nick repair

19 20-19 Polymerases II and III Pol II activity is not required for DNA replication Pol I appears mostly active in repair Only Pol III is required for DNA replication –Pol III is the enzyme that replicates bacterial DNA

20 20-20 The Pol III Holoenzyme Pol III core is composed of 3 subunits: –DNA polymerase activity is in the -subunit –3 5exonuclease activity found in -subunit –Not yet clear what is the role of -subunit DNA-dependent ATPase activity is located in the -complex containing 5 subunits Lastly, -subunit plus the other 8 comprise the holoenzyme

21 20-21 Fidelity of Replication Faithful replication is essential to life DNA replication machinery has a built-in proofreading system –This system requires priming –Only a base-paired nucleotide can serve as a primer for Pol III holoenzyme –If wrong nucleotide is incorporated accidentally replication stalls until 3 5 exonuclease of Pol III holoenzyme removes it Primers are made of RNA which may help mark them for degradation

22 20-22 Multiple Eukaryotic DNA Polymerases Mammalian cells contain at least 5 different DNA polymerases –Polymerases and appear to participate in replicating both DNA strands –Priming DNA synthesis is -subunit role –Elongating both strands is done by -subunit

23 20-23 Strand Separation DNA replication assumes that the 2 DNA strands at the fork somehow unwind This does not happen automatically as DNA polymerase does its job –2 parental strands hold tightly to each other –This takes energy and enzyme action to separate them –Helicase that unwinds dsDNA at the replicating fork is encoded by E. coli dnaB gene

24 20-24 Single-Strand DNA-Binding Proteins Prokaryotic ssDNA-binding proteins bind much more strongly to ssDNA than to dsDNA –Aid helicase action by binding tightly and cooperatively to newly formed ssDNA –Keep it from annealing with its partner By coating ssDNA, SSBs protect it from degradation SSBs are essential for prokaryotic DNA replication

25 20-25 Topoisomerases Strand separation of DNA is referred to as unzipping –DNA is not really like a zipper with straight, parallel sides, actually a double helix –When 2 strands of DNA separate, rotate around each other Helicase could handle this task alone if DNA were linear, short Closed circular DNA present special problems –As DNA unwinds at one site –More winding must occur at another site

26 20-26 Cairnss Swivel Concept A swivel in the DNA duplex called DNA gyrase Allows the DNA strands on either side to rotate to relieve the strain Gyrase belongs to the enzyme class topoisomerase These add transient single- or double-stranded breaks into DNA Serves to permit change in shape or topology

27 20-27 Topoisomerase Mechanism Enzymes called helicases use ATP energy to separate the two parental DNA strands at the replication fork As helicase unwinds 2 parental strands it introduces a compensating positive supercoiling force Stress of this force must be overcome or DNA will resist progression of replication fork This stress releasing mechanism is the swivel DNA gyrase acts as swivel by pumping negative supercoils into replicating DNA

28 DNA Damage and Repair DNA can be damaged in many different ways, if left unrepaired this damage can lead to mutation, changes in the base sequence of DNA DNA damage is not the same as mutation though it can lead to mutation If a particular kind of DNA damage is likely to lead to a mutation, we call it genotoxic

29 20-29 Definition of DNA Damage DNA damage is a chemical alteration –Mutation is a change in a base pair –Common examples of DNA damage Base modifications caused by alkylating agents Pyrimidine dimers caused by UV radiation

30 20-30 Damage Caused by Alkylation of Bases Alkylation is a process where electrophiles: –Encounter negative centers –Attack them –Add carbon-containing groups (alkyl groups)

31 20-31 Damage Caused by Alkylation of Bases Alkylating agents like ethylmethane sulfonate (EMS) add alkyl groups to bases –Some alkylation dont change base-pairing, innocuous –Others cause DNA replication to stall Cytotoxic Lead to mutations if cell attempts to replicate without damage repair –Third type change base-pairing properties of a base, so are mutagenic

32 20-32 Damage Caused by Radiation Ultraviolet rays –Comparatively low energy –Result in formation of pyrimidine dimers, also called cyclobutane pyrimidine dimers (CPDs) Gamma and x-rays –Much more energetic –Ionize molecules around the DNA –Form highly reactive free radicals that attack DNA Alter bases Break strands

33 20-33 DNA Damage: Pyrimidine Dimers and 8-oxoguanine

34 20-34 Directly Undoing UV DNA Damage UV radiation damage to DNA can be directly repaired by a photolyase, which is actually two separate enzymes that catalyze repair of CPDs Uses energy from near-UV to blue light to break bonds holding 2 pyrimidines together

35 20-35 Undoing High Energy DNA Damage O6 alkylations on guanine residues can be directly reversed by the suicide enzyme, O6- methylguanine methyltransferase This enzyme accepts the alkyl group onto the sulfur group of one of its cysteines and becomes irreversibly inactivated

36 20-36 Excision Repair Percentage of DNA damage products that can be handled by direct reversal is small Most damage involves neither pyrimidine dimers nor O6-alkylguanine Another repair mechanism is required, excision repair is the process that removes most damaged nucleotides –Damaged DNA is removed –Replaced with fresh DNA –Base and nucleotide excision repair are both used, BER and NER, respectively

37 20-37 Base Excision Repair Base excision repair (BER) acts on subtle base damage –Begins with DNA glycosylase Extrudes a base in a damaged base pair Clips out the damaged base Leaves an apurinic or apyrimidinic site that attracts DNA repair enzymes –DNA repair enzymes Remove the remaining deoxyribose phosphate Replace it with a normal nucleotide

38 20-38 Base Excision Repair in E. coli DNA polymerase I fills in missing nucleotide in BER Base is removed the AP site remains – apurinic or apyrimidinic AP endonuclease cuts or nicks DNA strand Phosphodiesterase removes the AP sugar phosphate Pol I performs repair synthesis

39 20-39 Eukaryotic BER DNA polymerase fills in the missing nucleotide –Makes mistakes –No proofreading activity APE1 carries out proofreading Repair of 8-oxyguanine (oxoG) sites in DNA is special case BER – can occur in 2 ways –A that has mispaired with oxoG can be removed after DNA replication by a specialized adenine DNA glycosylase –oxoG will still be paired with C and oxoG removed by another DNA glycoslyase, oxoG repair enzyme

40 20-40 Nucleotide Excision Repair Nucleotide excision repair typically handles bulky damage that distorts DNA double helix NER in E. coli begins when damaged DNA is clipped by an endonuclease on either side of the lesion, sites nt apart Allows damaged DNA to be removed as part of resulting base oligonucleotide

41 20-41 NER in E. coli Excinuclease (UvrABC) cuts either side Remove oligonucleotide nt DNA polymerase I fills in missing nucleotides using top strand as template DNA ligase seals the nick to complete the task

42 20-42 Eukaryotic NER Eukaryotic NER uses 2 paths GG-NER (global genome) –Complex composed of XPC and hHR23B initiates repair binding lesion in the genome –Causes limited amount of DNA melting –XPA and RPA are recruited –TFIIH joins, 2 subunits (XPB, XPD) use helicase to expand the melted region –RPA binds 2 excinucleases (XPF, XPG) positions for cleavage –Releases damaged fragment nt long

43 20-43 Congenital defects in DNA Repair Much of our information about repair mechanisms in humans has come from the study of congenital defects in DNA repair These repair disorders cause a group of human diseases, including Cockaynes syndrome and xeroderma pigmentosum (XP) Most XP patients are thousands of times more likely to develop skin cancer when exposed to the sun compared to healthy persons without XP

44 20-44 Transcription-Coupled NER TC-NER is very similar to GG-NER except: –RNA polymerase plays role of XPC in damage sensing and initial DNA melting In either type, DNA polymerase or fills in the gap left by removal of damaged fragment DNA ligase seals the DNA

45 20-45 Human Global Genome NER

46 20-46 Double-Strand Break Repair in Eukaryotes dsDNA breaks in eukaryotes are probably most dangerous form of DNA damage These are really broken chromosomes –If not repaired lead to cell death –In vertebrates can also lead to cancer Eukaryotes deal with dsDNA breaks in 2 ways: –Homologous recombination –Nonhomologous end-joining (NHEJ) Role of chromatin remodeling in dsDNA break repair

47 20-47 Model for Nonhomologous End-Joining This process requires Ku and DNA-PK cs which bind at DNA ends and lets ends find regions of microhomology 2 DNA-PK complexes phosphorylate each other and activates –Catalytic subunit to dissociate –DNA helicase activity of Ku to unwind DNA ends Extra flaps of DNA removed, gaps filled, ends permanently ligated

48 20-48 Role of Chromatin Remodeling in Double-Stranded Break Repair 2 protein kinases, Mec1 and Tel1, are recruited to DSBs They phosphorylate Ser129 of histone H2A in nearby nucleosomes Phosphorylation recruits chromatin remodeler IN080 to the DSB –Use DNA helicase activity to push nucleosomes away from DSB ends –Forms ssDNA overhangs essential for recombination SWR1 shares components with IN080 –Replaces phosphorylated H2A with variant Htz1

49 20-49 Mismatch Repair Mismatch repair system recognizes parental strand by methylated A in GATC sequence Corrects mismatch in progeny strand Eukaryotes use part of repair system Rely on different, uncharacterized method to distinguish strands at a mismatch

50 20-50 Coping with DNA Damage Without Repairing It Direct reversal and excision repair are true repair processes Eliminate defective DNA entirely Cells can cope with damage by moving around it –Not true repair mechanism –Better described as damage bypass mechanism

51 20-51 Recombination Repair The gapped DNA strand across from a damaged strand recombines with normal strand in the other daughter DNA duplex after replication Solves gap problem Leaves original damage unrepaired

52 20-52 Error-Prone Bypass Induce the SOS response This causes DNA to replicate even though the damaged region cannot be read correctly Result is errors in the newly made DNA

53 20-53 Error-Free Bypass in Humans Humans have relatively error-free bypass system that inserts dAMPs across from pyrimidine dimers Replicate thymine dimers correctly Uses DNA polymerase plus another enzyme to replicate a few bases beyond the lesion If DNA polymerase gene is defective, DNA polymerase and others take over

54 20-54 Error-Prone Bypass in Humans Errors in correcting UV damage lead to a variant form of XP, XP-V DNA polymerase is active on templates with thymidine dimers and AP sites The polymerase is not error-free With a gapped template, it is one of the least accurate template-dependent polymerases known

Download ppt "Molecular Biology Fifth Edition Chapter 20 DNA Replication, Damage, and Repair Lecture PowerPoint to accompany Robert F. Weaver Copyright © The McGraw-Hill."

Similar presentations

Ads by Google