Presentation on theme: "DNA Replication, Repair, and Recombination"— Presentation transcript:
1 DNA Replication, Repair, and Recombination Chapter 28DNA Replication, Repair, and Recombination
2 Outline DNA Replication is Semiconservative General Features of DNA ReplicationDNA PolymerasesThe Mechanism of DNA ReplicationEukaryotic DNA ReplicationTelomeres and TelomeraseDNA RepairReverse Transcriptase2
3 DNA ReplicationDouble Helix Facilitates the Accurate Transmission of Hereditary InformationSemiconservative replication:
4 Experiment of DNA semiconservative replication Meselson & Stahl ExperimentDensity-gradient equilibrium sedimentationParent DNA is labeled with 15N by growing E. Coli in 15N containing medium (15NH4Cl)Transfer E. Coli in 14N containing mediumLook at distribution
5 Significance of semiconservative replication The genetic information is transferred from one generation to the next generation with high fidelity.
6 DNA Replication: Melting of double helix Replication requires separation of the two strands of double helixHydrogen bonds between the base pairs are disruptedHeatAcid/alkaliInside a cell is done with the help of helicases which use ATPDissociation of double helix is termed as meltingIt occurs abruptly at a certain temperatureMelting temperature (Tm):Melting is monitored by measuring absorbance at 260 nm
7 DNA Replication: Melting of double helix The temperature at the midpoint of the transition (tm) is the melting point. It depends onpHionic strengththe sizebase composition of the DNAFIGURE 8-27a Heat denaturation of DNA. (a) The denaturation, or melting, curves of two DNA specimens. The temperature at the midpoint of the transition (tm) is the melting point; it depends on pH and ionic strength and on the size and base composition of the DNA.
8 DNA Replication: Melting of double helix Relationship between tm and the G+C content of a DNAFIGURE 8-27b Heat denaturation of DNA. (b) Relationship between tm and the G+C content of a DNA.
9 FIGURE 8-26 Reversible denaturation and annealing (renaturation) of DNA.
10 Annealing/hybridization DNA strands with similar sequences will form partial duplexes or hybrid with each other.Closer evolutionary relationshipbetween speciesSimilar DNA sequencesDNA hybridizeThis property is used to “fish out”(clone) a similar gene from differentspecies, if the gene sequence froma species is known.Why human DNA hybridizes much more extensively with mouse DNA than with yeast DNA?
11 DNA ReplicationReplication: polymerization of deoxyribonucleoside triphosphates along a templateWhat is required?
12 DNA Replication DNA Polymerase The first DNA Polymerase (short for DNA-pol I) was discovered in 1958 by Arthur Kornbergreceived Nobel Prize in physiology or medicine in 1959
13 Structure of DNA polymerase enzymes First determined DNA polymerase structure“Klenow fragment” of E. Coli DNA polymerase I
14 DNA Polymerases 5 structural classes Finger and thumb domains wrap around DNA and hold it across the enzyme’s active siteSimilar overall shapeSimilar mechanism
15 What DNA polymerases require for replication? TemplateDNA polymerase is a …………………………..that synthesizes a product with a base sequence complimentary to that of the templatePrimerDNA polymerase requires a primer with a free 3’-hydroxyl group already base- paired to the template.
16 Polymerase reaction Two bound metal ions participate in the reaction One metal ion attaches to dNTP and 3’-OH group of the primerSecond metal ion interacts only with dNTP.Two metal ions bridged by carboxylate groups of two Asp residues.
18 How accuracy is maintained during DNA replication? Binding of dNTP with correct base is favored by formation of a base pair with its partner on the template strandH-bonds contribute to this formationCan direct the incorporationof thymidineshape complimentarity
19 Why shape complementarity is important? First reason:Minor groove interactionsDNA polymerases donate 2H bonds to base pairs in minor grooveHydrogen bond acceptors are present in these 2 positions for all Watson-Crick base pairs
20 Why shape complementarity is important? Second reason:Shape selectivity:Binding of dNTP to DNA polymerase induces conformational changegenerates a tight pocketresidues lining this pocket ensure the efficiency and fidelity of DNA synthesis
21 Synthesis of RNA primer Primase: An RNA polymeraseSynthesizes a short stretch of RNAcomplimentary to one of the template DNA strandsLater removed by hydrolysis and replaced by DNA
22 How replication proceeds along the parent DNA? Both strands of parental DNA serve as templates.Site of DNA synthesis called “replication fork”.Parental DNA
23 How replication proceeds along the parent DNA? Unwinding of any single DNA replication fork proceeds in one directionProblemThe two DNA strands are of opposite polarity and DNA polymerases only synthesize DNA 5’ to 3’Solution: DNA is made in opposite directions on each templateLeading strand -synthesized 5’ to 3’ in the direction of the replication fork…………………..-requires a single RNA primerLagging strand -synthesized 5’ to 3’ in the opposite direction.-………………………-requires many RNA primersDNA is synthesized in short fragments called Okazaki fragments
24 How are Okazaki fragments joined? DNA ligase reaction:DNA ligase catalyzes formation of phosphodiester bondIn eukaryotes, this is and ATP-driven reactionIn bacteria, this is NAD-driven reactionDNA ligase seals breaks in dsDNA
25 FIGURE 25-16 Final steps in the synthesis of lagging strand segments FIGURE Final steps in the synthesis of lagging strand segments. RNA primers in the lagging strand are removed by the 5′→3′ exonuclease activity of DNA polymerase I and are replaced with DNA by the same enzyme. The remaining nick is sealed by DNA ligase. The role of ATP or NAD+ is shown in Figure
26 DNA ligase mechanismFIGURE Mechanism of the DNA ligase reaction. In each of the three steps, one phosphodiester bond is formed at the expense of another. Steps 1 and 2 lead to activation of the 5′ phosphate in the nick. An AMP group is transferred first to a Lys residue on the enzyme and then to the 5′ phosphate in the nick. In step 3, the 3′-hydroxyl group attacks this phosphate and displaces AMP, producing a phosphodiester bond to seal the nick. In the E. coli DNA ligase reaction, AMP is derived from NAD+. The DNA ligases isolated from some viral and eukaryotic sources use ATP rather than NAD+, and they release pyrophosphate rather than nicotinamide mononucleotide (NMN) in step 1 .
28 How are DNA strands separated? Helicases separate DNA strands for replicationHelicases utilizes energy of …………….to do soTypically oligomers with 6 subunitsEach subunit has P loop NTPase domainNeighboring subunits interact closely in the ring structureOnly a single strand of DNA can fit through the center of the ringDNA strand binds to loops on 2 adjacent subunits
29 Helicase Mechanism Initially both domains bind ssDNA Upon ATP binding, Cleft between domains closesA1 domain slides along DNAOn ATP hydrolysisCleft opens upPulls DNA from B1 domain toward A1dsDNA separated
30 DNA Unwinding and Supercoiling As helicase unwinds DNAthe DNA in front becomes overwoundtorsionally stressed DNA double helicesfold up on themselves to form tertiary structures
31 An electron micrograph showing negatively supercoiled and relaxed DNA TopoisomersCircular DNA molecules withsame nucleotide sequencedifferent linking numbersAn electron micrograph showing negatively supercoiled and relaxed DNA
32 Linking numberIt is equal to the number of times that a strand of DNA winds in the right-handed direction around the helix axis when the axis lies in a planeThe linking number for a relaxed B-DNA molecule:= the number of base pairs present/ 10.4………….. is the number of base pairs per turn
33 Other Terms Right-handed vs Left-handed Important numbers Linking number (Lk)Must be integerMolecules differing only in linking number are topoisomersTwisting number (Tw): a measure of the helical winding of DNA around each otherDoes not have to be integerWrithing number (Wr): a measure of the coiling of the axis of the double helix. i.e. supercoilingLk = Tw + Wr
35 Unwinding the linear duplex by two turns before joining its ends Two limiting conformations are possible:The DNA can fold into a structure containing 23 turns of B helix and an unwound loopThe double helix can fold up to cross itselfSuch crossings are called ……………..
36 Supercoiling Why is supercoiling biologically important? Supercoiled DNA has more compact shape (packaging becomes easy)Supercoiling affects DNA’s interactions with other molecules
37 Dealing with supercoiling during replication Negative supercoils must be removed and the DNA relaxed as the double helix unwindsTopoisomerases introduce or eliminate supercoilsType I TopoisomerasesCatalyze relaxation of supercoiled DNAType II TopoisomeraseAdds negative supercoils to DNA
38 Dealing with supercoiling during replication They alter the linking number of DNA in a 3-step processCleave one or both strandsType I cleaves one strandType II cleaves two strandsPassage of a segment of DNA through this breakReseal DNA break
39 Type I Topoisomerases Human type I topoisomerase comprises Four domains around a central cavityDiameter of 20 Å (diameter of B-DNA)Includes a tyrosine residue (Tyr 723)
40 Topoisomerase I Mechanism On binding to DNA, TopoI cleaves one strand of the DNA through a Tyr (Y) residue attacking a phosphate.When the strand is cleaved, it rotates in a controlled manner around the other strand.The reaction is completed by religation of the cleaved strand. This relaxes the DNA!
42 Type II Topoisomerases A more complex mechanismcuts dsDNAWill not be covered for Chem 361
43 Clinical importance of Types I and II topoisomerases Human topoisomerase IInhibited by Camptothecin, an antitumor agentBacterial topoisomerase II (DNA gyrase)Target of several antibioticsNovobiocin blocks binding of ATP to gyraseNalidixic acid and ciprofloxacin interfere with breakage and rejoining of DNA chainsUsed to treat urinary track and other infectionsIncluding Bacillus anthracis (anthrax)
44 DNA Replication is Highly Coordinated Coordination of enzyme activity is required for precise and rapid replication of genome. -Requires highly processive polymerases : Example: DNA Pol IIIStructure of sliding clampIt allows the polymerase to move with DNA
45 The leading and lagging strands are synthesized in a coordinated fashion DNA polymerase III synthesizes
46 The leading and lagging strands are synthesized in a coordinated fashion DNA-poly III begins synthesis of the leading strand starting from RNA primerHelicase unwinds DNAss-binding proteins bind to the unwound strands, keeping the strands separated so that both strands can serve as templatesLagging synthesis more complexDNA-poly III makes Okazaki fragmentsDNA-poly I removes ………………….DNA ligase connects fragmentsDNA synthesis in eukaryotes, more complex
47 The leading and lagging strands are synthesized in a coordinated fashion DNA-poly III begins synthesis of leading strand using RNA primerHelicase unwinds DNAss-binding proteins keep strands separated so both can be templates.Lagging strand synthesis more complexLagging strand
48 The leading and lagging strands are synthesized in a coordinated fashion The mode of synthesis of the lagging strand is more complexLagging strand is synthesized in fragmentssuch that 5′ → 3′ polymerization leads to overall growth in the 3′ → 5′ directionYet the synthesis of the lagging strand is coordinated with the synthesis of the leading strand
49 The leading and lagging strands are synthesized in a coordinated fashion How is this coordination accomplished?DNA polymerase IIIThe holoenzyme includes two copies of the polymerase core enzymeThe core enzymes are linked to a central structure having the subunit composition γτ2δδ′χφThe entire apparatus interacts with the hexameric helicase DnaB
50 The leading and lagging strands are synthesized in a coordinated fashion Okazaki fragments (RNA polymerase initiates)Looping the template for the lagging strand places it in position for 5’--->3’ polymerizationDNA poly III lets go off the lagging strand after adding 1000 nucleotidesNew loop formedRNA primer made by primaseGaps filled by ……………….(it removes primers too)
51 Prokaryotes: Replication Origin of ReplicationIn E. coli: a unique site “origin of replication” is called oriC locus
52 Prokaryotes: Replication The binding of DnaA molecules to one another signals the start of the preparatory phaseThe DnaA proteins bind to the five high-affinity sites in oriCDnaA molecules form an oligomera cyclic hexamerThe DNA is wrapped around the outside of the DnaA hexamer
53 Prokaryotes: Replication Single DNA strands are exposed in the prepriming complexmakes single-stranded DNA accessible to other proteinsDnaAoriC:Preparation for replicationDnaB (hexameric helicase) + DnaC (helicase loader)SSB“Prepriming complex”DnaG (primase) inserts the RNA primer
54 Prokaryotes: Replication The polymerase holoenzyme assemblesDNA pol III holoenzyme +Prepriming complexATP hydrolysis within DnaABreakup of DnaA(prevents additional round of replication!)
55 Eukaryote oriC is more complex Eukaryotes: ReplicationEukaryote oriC is more complexE. ColiHuman diploid cellReplicates 6 billion bp23 pairs of chromosomes must be replicatedLinear chromosomeReplicates 4.6 million bpGenetic information contained in 1 chromosomeCircular chromosome
57 Greatest replication problem with linear chromosomes Complete replication of DNA ends is difficultpolymerases act only in the 5′ → 3′ directionthe lagging strand would have an incomplete 5′ end after the removal of the RNA primereach round of replication would further shorten the chromosome
58 Telomers (from Greek: telos = end) Ends of chromosomes are differentHundreds of tandem repeats of six-nucleotide sequenceOne of the strands is G rich at the 3′ end, and it is slightly longer than the other strandProposed modelSingle-stranded region invades duplex to form large duplex loop
59 Telomeres are replicated by telomerase, a specialized polymerase that carries its own RNA template contains an RNA molecule that serves as the template for the elongation of the G-rich strandcarries the information necessary to generate the telomere sequences
60 DNA Damage DNA does become damaged in the course of replication through other processesDamage to DNA can besimpleas the misincorporation of a single basecomplexchemical modification of baseschemical cross-links between the two strands of the double helixbreaks in one or both of the phosphodiester backbonesResultscell death or cell transformationchanges in the DNA sequence that can be inherited by future generationsblockage of the DNA replication process itself
61 Sources of Damage: Bases can be damaged by oxidizing agents, alkylating agents, and light Oxidation: Reactive oxygen specieshydroxyl radical reacts with guanine to form 8-oxoguanine8-Oxoguanine is mutageneticDeamination: potentially deleterious processadenine can be deaminated to form hypoxanthinemutagenichypoxanthine pairs with cytosine rather than thymineOxidationDeamination
62 Sources of Damage: Bases can be damaged by oxidizing agents, alkylating agents, and light Alkylation:Electrophilic centers can be attacked by nucleophilesN-7 of guanine and adenine form alkylated adductsAflatoxin B1produced by molds that grow on peanuts and other foodsconverted into a highly reactive epoxide by a cytochrome P450 enzymereacts with the N-7 atom of guanosine to form a mutagenic adduct that frequently leads to a G–C-to-T–A transversion
63 Sources of Damage: Bases can be damaged by oxidizing agents, alkylating agents, and light Ultraviolet light: ubiquitous DNA-damaging agentcovalently links adjacent pyrimidine residues along a DNA strandpyrimidine dimer cannot fit into a double helixblocks replication and gene expressionA thymine dimer is an example of an intrastrand cross-linkCross-links between bases on opposite strands also can be introduced by various agents
64 Sources of Damage: Bases can be damaged by oxidizing agents, alkylating agents, and light High-energy electromagnetic radiation: X-raysproduces high concentrations of reactive species in solutioninduces several types of DNA damagesingledouble-stranded breaks in DNA
65 DNA damage can be detected and repaired by a variety of systems DNA repair pathways:Mismatch repair: correction in placeNucleotide excision repair: a stretch of DNA is removedBase excision repair: damaged base is removed and replaced
66 TABLE 25-5 Types of DNA Repair Systems in E. coli
67 Error correction by the 3′→5′ exonuclease activity of DNA polymerase DNA polymerasesable to correct many DNA mismatches produced in the course of replicationthe ε subunit of E. coli DNA polymerase III functions as a 3′-to-5′ exonuclease
68 Error correction by the 3′→5′ exonuclease activity of DNA polymerase DNA polymerasesAs a new strand of DNA is synthesized, it is proofreadincorrect base slows down DNA synthesisdifficulty of threading a non-Watson–Crick base pair into the polymerasemismatched base is weakly boundable to fluctuate in positionslowdown allows time for these fluctuations to take the newly synthesized strand out of the polymerase active site and into the exonuclease active sitethe DNA is degraded, one nucleotide at a time, until it moves back into the polymerase active site and synthesis continues
69 Exonuclease activity ahead of the polymerase activity Error correction by the 3′→5′ exonuclease activity of DNA polymeraseExonuclease activity ahead of the polymerase activityA mismatched base (here, a C–A mismatch) impedes translocation of DNA polymerase I to the next site.Sliding backward, the enzyme corrects the mistake with its 3′→5′ exonuclease activity, then resumes its polymerase activity in the 5′→3′ direction.Contributes to remarkable fidelity of DNA replication with an error rate of less than ……………………….FIGURE 25-7 An example of error correction by the 3′→5′ exonuclease activity of DNA polymerase I. Structural analysis has located the exonuclease activity ahead of the polymerase activity as the enzyme is oriented in its movement along the DNA. A mismatched base (here, a C–A mismatch) impedes translocation of DNA polymerase I to the next site. Sliding backward, the enzyme corrects the mistake with its 3′→5′ exonuclease activity, then resumes its polymerase activity in the 5′→3′ direction.
70 Mismatch-repair systems Mismatch-repair systems consist of at least two proteinsMutSfor detecting the mismatchMutLfor recruiting an endonuclease (MutH)cleaves the newly synthesized DNA strand close to the lesion to facilitate repair
71 Direct Repair Example: photochemical cleavage of pyrimidine dimers Nearly all cells contain a photoreactivating enzyme called DNA photolyaseThe enzyme binds to the distorted region of DNAUses light energythe absorption of a photon by the N5,N10-methenyltetrahydrofolate coenzyme forms an excited statecleaves the dimer into its component bases
74 Nucleotide Excision repair: T-dimer is repaired by three enzymes:Excinucleasedetects the distortion and then cuts the damaged DNA strand at two sites8 nucleotides away from the damaged site on the 5′ side4 nucleotides away on the 3′ side.2. DNA polymerase IFor repair synthesis3. DNA ligase
75 Uracil Repair U formed by the deamination of C excised and replaced by C!!
76 Why is T instead of U in DNA? T or U pairs with AOnly difference: a methyl group in TC in DNA spontaneously deaminates forming U (100 events per day!, deamination of A and G much slower)Potentially mutagenic because U-A occurs rather than C-GPrevented by DNA glycosidaseThis enzyme cuts U but does not attack Tthe -CH3 in T is a tag that distinguishes T from deaminated C
77 Defective repair of DNA--->Cancer Xeroderma pigmentosum (AR)Extreme sensitivity to UVSkin is dryKeratosisSkin cancerDeath before the age of 30!Defect: Excinuclease part of repair system
78 Mutagen Detection Many human cancers caused by chemicals! Chemicals usually cause mutationsHow do we identify them?Bruce Ames developed a simple, sensitive test called “Ames” for detecting chemical mutagens.0.5 microg of 2-aminoantharacene gives 11,000 colonies; only 30 colonies in its absence!
79 Ames testThin layer of agar with salmonella bacteria cannot synthesize HisAddition of chemical mutagen to center results in many mutations (one making bacteria synthesize His again)Revertants make many coloniesWhat if P-450 is involved in mutagenesis?
80 FIGURE 25-21 Ames test for carcinogens, based on their mutagenicity FIGURE Ames test for carcinogens, based on their mutagenicity. A strain of Salmonella typhimurium having a mutation that inactivates an enzyme of the histidine biosynthetic pathway is plated on a histidine-free medium. Few cells grow. (a) The few small colonies of S. typhimurium that do grow on a histidine-free medium carry spontaneous back-mutations that permit the histidine biosynthetic pathway to operate. Three identical nutrient plates (b), (c), and (d) have been inoculated with an equal number of cells. Each plate then receives a disk of filter paper containing progressively lower concentrations of a mutagen. The mutagen greatly increases the rate of back-mutation and hence the number of colonies. The clear areas around the filter paper indicate where the concentration of mutagen is so high that it is lethal to the cells. As the mutagen diffuses away from the filter paper, it is diluted to sublethal concentrations that promote back-mutation. Mutagens can be compared on the basis of their effect on mutation rate. Because many compounds undergo a variety of chemical transformations after entering cells, compounds are sometimes tested for mutagenicity after first incubating them with a liver extract. Some substances have been found to be mutagenic only after this treatment.
83 Double stranded DNA molecules with similar sequences sometimes recombine DNA replication copies genetic messages as faithfully as possibleSeveral biochemical processes require recombination of genetic material between two DNA moleculesRecombination plays important role inMaking molecular diversity for AbManipulating genesGeneration of “gene knockout mice”
84 RECOMBINATION: two DNA molecules can recombine to form new DNA molecules with segments fromboth parental molecules
86 The genes of some viruses are made of RNA Reverse TranscriptionGenes in all pro and eukaryotes made of DNAIn viruses, genes made of DNA or RNARNA is like DNA but:Sugar is riboseU instead TRNA can be single or double stranded
87 The genes of some viruses are made of RNA Reverse Transcription Genetic info of RNA virus is contained in its RNAExample: Tobacco mosaic virus which infects tobacco plantsIt consists of a single strand of RNA surrounded by a protein coatAn RNA-directed RNA polymerase copies the viral RNAInfected cells die as the virus instructs the cell to commit suicide and results in discoloration of tobacco leafExample: RetrovirusKnown as retroviruses because information flows BACKWORD (RNA---DNA )not from DNA--- RNA.Includes HIV-1 as well as a number of RNA viruses that produce tumors
88 Reverse Transcription Synthesis of ssDNA complementary to ssRNA, forming a RNA-DNA hybrid.Hydrolysis of ssRNA in the RNA-DNA hybrid by RNase activity of reverse transcriptase, leaving ssDNA.Synthesis of the second ssDNA using the left ssDNA as the template, forming a DNA-DNA duplex.
89 Viral infection of RNA virus Reverse TranscriptionViral infection of RNA virusViral DNA gets incorporated into the chromosomal DNA of the host and is replicated along with the normal DNALater the integrated viral genome is expressed to form viral RNA and viral proteins