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 radiation • If uncorrected, such changes may interfere with the ability of the cell to function • DNA damage can be repaired by several mechanisms • All carcinogens cause changes in the DNA sequence and thus DNA damage and repair are important aspects in the development of cancer • Prokaryotic and eukaryotic DNA-repair systems are analogous
(transition) (transversion) purine pyrimidine Replication errors and their repair The nature of mutations: Point mutation 1. Switch of one base for another: 2. insertion or deletion of a nucleotide
Drastic changes in DNA Deletion Insertion Rearrangement of chromosome By insertion of a transposon, or aberrant actions of recombination Process.
Mismatch repair removes errors escape proofreading 2. The system must correct the mismatch accurately. 1. It must scan the genome. Scan DNA MutL activate MutH Distortion in the backbone Embracing mismatch; Inducing a kick in DNA; Conformational change in MutS itself Nicking is followed by Helicase (UvrD) and one of exonucleases (III)
DNA methylation to recognize the parental strain Once activated, MutH selectively nicks the Unmethylated strand.
Mismatch repair system in Eukaryotics MutS MutL E. coli MSH (MutS homolog) MLH or PMS Eukaryotics Hereditary nonpolyposis colorectal cancer (mutations in human homologes of Muts and MutL)
DNA damage Radiation, chemical mutagens, and spontaneous damage spontaneous damage due to hydrolysis and deamination deamination Base pair with A depurination
DNA damage spontaneous damage to generate natural base deamination Methylated Cs are hot spot for spontaneous mutation in vertebrate DNA
Base deamination leads to the formation of a spontaneous point mutation
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.
DNA damage by UV Thymine dimer These linked bases are incapable of base-pairing and cause DNA polymerase to stop.
Mutations caused by base analogs and intercalating agents Base analogs Thymine analog Analogs mispair to cause mistakes during replication
Mutations caused by intercalating agents Intercalating agents flat molecules Causing addition or deletion of bases during replication
Chemical carcinogens react with DNA and the carcinogenic effect of a chemical correlates with its mutagenicity
Aflatoxin can lead to a modification of guanosine (in tobacco smoke)
DNA damage by UV light The 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 dimer a, cyclobutane dimer (most lethal form) b, 6-4 photoproduct (most mutagenic form) Both DNA lesions are bulky and distort the double helix The thymine dimers block transcription and replication, and are lethal unless repaired.
UV survival curves The 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 – PHOTOREACTIVATION UV-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
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 by E. coli genes phrA and phrB. This enzyme binds to pyrimidinedimers 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).
Direct reversal of DNA damage photoreactivation Capture energy from light breaking covalent bond
Dark repair or light independent mechanisms • 3 mechanisms: • Excision repair – removal of damaged DNA strand followed by DNA synthseis • Recombinational repair - using other duplexes for repair. • SOS error-prone ‘repair’ – tolerance of DNA damage
Dark repair processes are defined by mutations in key genes uvrA, uvrB, uvrC, uvrD - excision repair recA, recB, recC - recombination, recA, - SOS error-prone repair polA (DNA pol I) All are very sensitive to UV light uvrA-recA- mutants are totally defective at dark repair and are killed by the presence of just one pyrimidine dimer
Excision repair In 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.
Base excision repair If a damaged base is not removed by base excision before DNA replication: a fail-safe system oxoG:A repair
T T Excision Repair in E.coli Damage recognised by UvrABC, nicks made on both sides of dimer 5’ 3’ 3’ 5’ T T 5’ 3’ 3’ 5’ T T Dimer removed by UvrD, a helicase 3’ 5’ 5’ 3’ Gap filled by DNA pol I and the nick sealed by DNA ligase 3’ 5’ 5’ 3’
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)
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)
Homologous DNA recombination RecA protein is essential for homologous recombination (from T.A.Brown. Genetics a molecular approach)
Summary Both 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) repair This error –prone repair mechanism is a last resort to ensure survival