Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32Slide 33Slide 34Slide 35Slide 36DNA Repair• DNA synthesis• proofreading• errors in DNA replication• mismatch repair• spontaneous mutations• base excision repairDNA Rearrangements• bacterial transposons• retrotransposition• viral life cyclesReferences: p. 198-199, 205-207, 211-216, 221-226Chapter 6DNA replicationA strand of DNA serves as a template for the synthesis of a complementary antiparallel strand in the ______ direction.DNA replicationFigure: p. 204The formation of thephosphodiester bondThe breaking of thehigh-energy Phosphoanhydride bond (*) in incomingdNTPs provides theenergy required forformation of the newphosphodiester bond by ___DNA POLYM!!!!!___________.Proofreading during DNA synthesisDNA polymerase also has 3’ to 5’ exonuclease activity• if the wrong nucleotide is added during 5’ to 3’ DNA synthesis, DNA polymerase can remove it via its 3’ to 5’ exonuclease activity• the enzyme then replace it with the correct nucleotide by resuming the 5’ to 3’ polymerase activityProofreading during DNA synthesisFor DNA polymerase to be able to proofread, DNA must be synthesized in the 5’ to 3’ direction.• during a hypothetical 3’ to 5’ DNA polymerization, the energy required for formation of the phosphodiester bond would come from hydrolyzing the high-energy phosphoanhydride bond at the 5’ end of the chain, But:• if the 5’ nucleotide (with its triphosphate) were removed as part of proofreading, no high-energy bond would be available for the addition of the correct nucleotideProofreading during DNA synthesisProofreading duringDNA synthesisThe consequence of errors in DNA replication is nucleotidemismatches that can lead to permanent changes if they are leftunrepaired before the next round of DNA synthesis.Errors in DNA replicationConsequences of errors in DNA replicationC => AG => TConsequences of errors in DNA replicationG => TC => AC => AConsequences of errors in DNA replicationC => AMismatch repair The wrong nucleotide is added by mistake during DNAreplication and needs to be replaced.Problem: How does the cell know which strand is the newlysynthesized strand (and hence, which one of the twonucleotides in a mismatched pair is the error?)Solution: The newly synthesized strand has breaks (nicks). replication of the new strand is not continuousDNA mismatch repair proteins must correct the error before DNA ligase seals the nickDiscontinuousDNA replicationFigure: p. 205• DNA mismatch repair proteinsrecognize the distortion due to mismatched pair & bind• the DNA is scanned for a nearby nick• the nicked strand is digestedfrom the nick back to the mismatch site• DNA polymerase and DNA ligase complete the repairModel for mismatch repair in eukaryotesDefects in mismatch repair proteinsMutS and MutL encode DNA mismatch repair proteins• Defects in these genes results in higher frequency of mutations in other genes, such as those that regulate cell proliferation= predispose an individual to cancerHereditary nonpolyposis colorectal cancer (HNPCC)DNA can be altered by interacting with other molecules in the cell or by UV-radiation, resulting in chemical changes.Spontaneous mutationsCategories of spontaneous mutations:• deamination• depurination• radiation-induced dimerizationConsequences:• distorted strands• mismatches that include unusual bases.DeaminationConsequences of cytosine deaminationC => UG => ADepurinationDepurination results in: • an apurinic site• no base pairingConsequence of depurinationRadiation-induced mutationUV-induced thymine dimer• a cyclobutane ring forms between adjacent thymine bases• the joined thymines can not pair with adenines• this is the basis for UV decontamination1) Direct reversal of DNA damage2) Base excision repair1) Direct reversal (direct repair):• repairs the altered molecule by reversing the chemicaltransformation• requires specific enzymes for each individual lesionExample: some organisms can reverse thymine dimers byusing a specific photoreactivating enzyme (photolyase)General categories of repair mechanisms forspontaneous mutationsRadiation-induced mutationBase excision repair2) Base excision repair is a general repair mechanism fornucleotide damage. NOT IMPROPER LINKING • the chemical transformation is not reversed• instead, the damaged base(s) are replaced- the unpaired base is recognized as a distortion and replaced before the next round of DNA replicationDNA glycosylase removes the base, an endonucleaserecognizes the site and cleaves the phosphodiester bond,and a deoxyribosephosphodiesterase removes theremaining sugar and phosphate from the site.Base excision repairSTEP 1STEP 2STEP 3DNA polymerase adds a new nucleotide.DNA ligase seals the nick in the backbone.Base excision repairXeroderma pigmentosum:Autosomal recessive genetic disorderBacterial mobile genetic elementsInsertion sequences: simple bacterial transposonsIR IR(Recombination enzyme)(Inverted repeats)Bacterial transposons may carry antibiotic resistance genes.(Ampicillin resistance gene)Insertion of simple bacterial transposonsNonreplicative transposition:• the enzyme transposase cleaves the insertion sequenceat the ends of the inverted repeats of the donor site• transposase also cleaves a random target site• the insertion sequence is “cut” from the donor site and “pasted” into the target siteResult: the insertion sequence moves from one site in thegenome to anotherReplicative transposition:• a copy of the insertion sequence is made by local DNAreplication and pasted into the target siteResult: a new copy of the insertion sequence appears at thetarget site with the original insertion sequence intactInsertion of simple bacterial transposonsRetrotransposition is catalyzed by reverse transcriptase (RT) synthesizing a DNA copy of the transcribed retrotransposon.• retrotransposons are unique to eukaryotes• the process can generate multiple copies of the original mobile element• in some plants, retrotransposons account for most of the DNA in the genomeRetrotranspositionRetrotranspositionintegraseDNA virusinfection cycleRetroviralinfection cyclelipid bilayer+ glycoproteinproteinRetroviralinfection