2 DNA damage and repair and their role in carcinogenesis A DNA sequence can be changed by copying errors introduced by DNA polymerase during replication and by environmental agents such as chemical mutagens or radiationIf uncorrected, such changes may interfere with the ability of the cell to functionDNA damage can be repaired by several mechanismsAll carcinogens cause changes in the DNA sequence and thus DNA damage and repair are important aspects in the development of cancerProkaryotic and eukaryotic DNA-repair systems are analogous
7 Mismatch repair removes errors escape proofreading 1. It must scan the genome.2. The system must correct the mismatch accurately.Scan DNADistortion in the backboneMutL activate MutHEmbracing mismatch;Inducing a kick in DNA;Conformational change inMutS itselfNicking is followed by Helicase (UvrD) and one of exonucleases(III)
8 DNA methylation to recognize the parental strain Once activated,MutH selectively nicks theUnmethylated strand.
10 Mismatch repair system in Eukaryotics MutSMutLE. coliMSH(MutS homolog)MLH or PMSEukaryoticsHereditary nonpolyposis colorectal cancer(mutations in human homologes of Muts and MutL)
11 DNA damage Radiation, chemical mutagens, and spontaneous damage spontaneous damage due to hydrolysis and deaminationdeaminationBase pair with Adepurination
12 DNA damage spontaneous damage to generate natural base deaminationMethylated Cs are hot spot for spontaneous mutation in vertebrate DNA
13 Base deamination leads to the formation of a spontaneous point mutation
14 Damaged by alkylation and oxidation Alkylation at the oxygen of carbon atom 6 of G : O6-metylguanine,often mispairs with T.Oxidation of G generates oxoG, it can mispair with A and C. a G:C to T:A transversion is one of the most common mutation in human cancers.
15 DNA damage by UV Thymine dimer These linked bases are incapable of base-pairing and causeDNA polymerase to stop.
16 Mutations caused by base analogs and intercalating agents Thymine analogAnalogs mispair to cause mistakes during replication
17 Intercalating agents Mutations caused by intercalating agents flat moleculesCausing addition or deletion of bases during replication
18 Chemical carcinogens react with DNA and the carcinogenic effect of a chemical correlates with its mutagenicity
19 modification of guanosine Aflatoxin can lead to amodification of guanosine(in tobacco smoke)
20 DNA damage by UV lightThe killing spectrum of UV light coincides with the peak absorbance of DNA for UV light, suggesting that DNA is the key macromolecule that is damaged.UV light causes dimerization of 2 adjacent pyrimidine (thymines).There are 2 forms of the dimera, cyclobutane dimer (most lethal form)b, 6-4 photoproduct (most mutagenic form)Both DNA lesions are bulky and distort the double helixThe thymine dimers block transcription and replication, and are lethal unless repaired.
22 UV survival curvesThe UV survival curve for both mutant and wild-type indicates that there are repair systems to deal with UV –damaged induced DNA.2 key observations:UV-irradiated bacteria if exposed to visible light showed an increased survival relative to those not exposed to visible light – PHOTOREACTIVATIONUV-irradiated bacteria if held in non-nutrient buffer for several hours in the dark, also showed enhanced survival relative to controls which had not – LIQUID HOLDING RECOVERY or DARK REPAIR
23 Photoreactivation repair The enhanced survival of UV-irradiated bacteria following exposure visible light is now known to be due to PHOTOLYASE, an enzyme that is encoded byE. coli genes phrA and phrB.This enzyme binds to pyrimidine dimers and uses energy from visible light (370 nm) to split the dimers apart.Phr- mutants were defective at photoreactivation.Similar enzymes are found in other bacteria, plants and eukaryotes (but not present in man).
24 (from T.A.Brown. Genetics a molecular approach)
25 Direct reversal of DNA damage photoreactivationCapture energy from lightbreakingcovalent bond
26 Dark repair or light independent mechanisms Excision repair – removal of damaged DNA strand followed by DNA synthseisRecombinational repair - using other duplexes for repair.SOS error-prone ‘repair’ – tolerance of DNA damage
27 Dark repair processes are defined by mutations in key genes uvrA, uvrB, uvrC, uvrD - excision repairrecA, recB, recC recombination,recA, - SOS error-prone repairpolA (DNA pol I)All are very sensitive to UV lightuvrA-recA- mutants are totally defectiveat dark repair and are killed by the presence ofjust one pyrimidine dimer
28 Excision repairIn this form of repair the gene products of the E. coli uvrA, uvrB and uvrC genes form an enzyme complex that physically cuts out (excises the damged strand containing the pyrimidine dimers.An incision is made 8 nucleotides (nt) away for the pyrimidine dimer on the 5’ side and 4 or 5 nt on the 3’ side.. The damaged strand is removed by uvrD, a helicase and then repaired by DNA pol I and DNA ligase.Is error-free.
29 Base excision repairIf a damaged base is not removed by base excision before DNA replication: a fail-safe systemoxoG:A repair
30 Excision Repair in E.coli Damage recognisedby UvrABC, nicksmade on both sides ofdimer5’3’3’5’TT5’3’3’5’TTDimer removed by UvrD, a helicaseT3’5’5’3’Gap filled by DNA pol I and the nick sealed by DNA ligase3’5’5’3’
32 Excision repair The UvrABC complex is referred to as an exinuclease. UvrAB proteins identify the bulky dimer lesion, UvrA protein then leaves, and UvrC protein then binds to UvrB protein and introduces the nicks on either side of the dimer.In man there is a similar process carried out by 2 related enzyme complexes: global excision repair and transcription coupled repair.Several human syndromes deficient in excision repair, Xeroderma pigmentosum, Cockayne Syndrome, and are characterised by extreme sensitivity to UV light (& skin cancers)
33 Base excision repair NOT a major form of repair of UV-induced DNA damage, but an important form of DNA repair generally.(from T.A.Brown. Genetics a molecular approach)
34 RecA protein is essential for homologous recombination DNA recombinationRecA protein is essential for homologous recombination(from T.A.Brown. Genetics a molecular approach)
35 SummaryBoth the dark repair mechanisms and photo-reactivation are very accurate and can deal with low levels of DNA damage.However, extensive damage levels to elevated levels of excision and recombinational repair, and also the activation of another repair system which is error-prone (SOS) repairThis error –prone repair mechanism is a last resort to ensure survival