分子生物學：第6章 DNA損傷與修復

1、 DNA損傷與修復 DNA DAMAGE AND REPAIR Deoxyribonucleic Acid （DNA）NNNHNNH2lAdenine NHNNHNONH2lGuanineConstitution of DNAuracilNHNHOONNHNH2OcytosineNHNHOOthymine ribose (deoxyribose) + base ribose (deoxyribose) + base C-N bond C-N bond(OH)(OH)Nucleotide（核苷酸）（核苷酸） O-POO-NNNNNH2OHHOHHOHHOCH2O-POO-O-POO-三磷酸腺苷

2、(ATP)AMPADPATPd AMP 5533DNA : poly nucleotides DNA Damage DNA 損傷的原因和后果損傷的原因和后果 DNA自發(fā)性損傷：DNA復制和自發(fā)性變化 物理因素 化學因素 生物因素DNA復制誤差核苷酸錯誤摻入： 摻入率 為10-10 DNA聚合酶使用核酸外切酶活性 C A堿基錯配堿基錯配自主突變堿基異構突變：核苷酸上堿基互換脫氨基：環(huán)外氨基脫落C變成U，A變成H,G變成X。脫嘌呤和脫嘧啶：嘌呤和嘧啶水解堿基修飾與斷鏈：自由基CH3、O2 和細胞代謝物 能修飾DNA或產生單雙鏈斷裂Single base change Deamination（去氨基

3、）（去氨基） C changes into U，U-G became C-G.Depurination The bond linking base to sugar weekened, is easy to break.物理因素引起的DNA損傷 紫外線 DNA被小于260nm波長紫外線照射時，形成嘧啶二聚體。也能產生DNA斷裂。TT， CC， TC 二聚體 1.Ultraviolet irradiation A- T became T-TAT互換互換電離輻射引起DNA損傷 電離輻射產生電離輻射的直接和間接作用，導致堿基改變、互換，脫落或修飾、脫氧核糖變化、DNA單雙鏈斷裂 或交聯(lián)（DNA-DN

4、A,DNA-蛋白質）。電離輻射的直接和間接作用DNA鏈斷裂鏈斷裂化學因素引起的DNA損傷氧化劑：氧化劑： 8-oxo-7 8-dihydroguanine (8-oxoG) Generation of DNA strand interruptions from reactive oxygen species.化學因素引起的DNA損傷烷化劑：烷化劑： 一類親電子化合物，很容易和一類親電子化合物，很容易和DNA發(fā)生化學反發(fā)生化學反應，應， 產生堿基烷基化、脫落、斷鏈或交聯(lián)。產生堿基烷基化、脫落、斷鏈或交聯(lián)。Alkylation（烷基化）（烷基化） -CH3 was added to DNA bas

5、es.Viruses The replication of damaged DNA before cell division can lead to the incorporation of wrong bases opposite damaged ones. Daughter cells that inherit these wrong bases carry mutations from which the original DNA sequence is unrecoverable (except in the rare case of a back mutation, for exam

6、ple, through gene conversion).生物因素生物因素DNA損傷的后果損傷的后果點突變點突變缺失缺失插入插入到位或轉座到位或轉座斷裂斷裂* 突變可以是進化上的需要。有意義，無意義。DNA RepairDNA repair Direct repair Excision repair Mismatch repair Recombination repair SOS system1. Direct Reversal of Base Damage2. Excision Repair1. Base Excision Repair (BER)2. Nucleotide Excisio

7、n Repair (NER)3. Mismatch Repair (MMR)Direct repair Photoreactivation Photreactivation enzyme dialyzes T-T dimer by light, leading to repair of DNA. Methyl transferase Remove -CH3 and get complete DNA. NNOOCH3RPNNOOCH3RNNOOCH3RPNNORUVOCH3胸嘧啶二聚體( T T )Photolyase1) Remove damaged region2) Resynthesis

8、（再合成）DNA 3) Ligation（結扎） BER Special enzymes replace just the defective（錯誤的） base 1 snip（減去） out the defective base 2 cut the DNA strand（一段） 3 Add fresh nucleotide 4 Ligate gap A base-specific DNA glycosylase detects an altered base and removes itAP endonuclease and phosphodiesterase remove sugar ph

9、osphate.DNA Polymerase fills and DNA ligase seals the nickNucleotide Excision repair（NER）With the help of some endonucleases（核酸內切酶）, DNA was cut away and new DNA was synthesized. Same as Base Excision Except that It recognizes more varieties of damage Remove larger segments of DNA (10 -100s of bases

10、) a large multienzyme compound scans the DNA strand for anomalities upon detection a nuclease cuts the strand on both sides of the damage DNA helicase removes the oligonucleotide the gap is repaired by DNA polymerase and DNA ligase enzymesMismatch repairDuring the replication of DNA, wrong bases wer

11、e input, Mismatch repair enzymes recognize this and cut the wrong part.Mismatch repair systemDNA methylaseDNA polymerase Helicase SSB Endonuclease LigaseMCE (mismatch correct enzyme) 3 subunits mut H, L, S Mismatch repair deals with correcting mismatches of the normal bases; that is, failures to mai

12、ntain normal base pairing (AT, C G) Recognition of a mismatch requires several different proteins including one encoded by MSH2. Cutting the mismatch out also requires several proteins, including one encoded by MLH1. In E. coli, certain adenines become methylated shortly after the new strand of DNA

13、has been synthesized. The MMR system works more rapidly, and if it detects a mismatch, it assumes that the nucleotide on the already-methylated (parental) strand is the incorrect one and removes the nucleotide on the freshly-synthesized daughter strand. How such recognition occurs in mammals is not

14、yet known. The assumed mechanisms for mismatch repair. Mismatches are detected by a dimer of the mismatch repair protein MutS (encoded by MSH2). MutL (encoded by MLH1) has weak ATPase activity and it is also shown to be a DNA endonuclease same as MutH. The entire MutSHL complex then slides along the

15、 DNA in the direction of the mismatch, liberating the strand to be excised as it goes. An exonuclease trails the complex and digests the ss-DNA tail This type of repair is much more complicated than is excision repair, and requires many more gene products. The products of a number of these repair ge

16、nes are induced by radiation damage, and therefore this type of repair requires protein synthesis before it can function. Because of its complexity, this type of repair makes mistakes(a) The dots indicate lesions in the DNA.(b) DNA synthesis proceeds up to a lesion and then skips past the lesion, le

17、aving a gap in the daughter strand. (c) Filling of the daughter strand gaps with DNA from parental strands by a recombinational process that requires a functional recA gene.(d) Gaps in the parental strands are repaired by repair replication This process occurs in the part of the chromosome that was

18、replicated prior to irradiation, i.e., where two sister duplexes were present before irradiation. After the excision of the lesion, the resulting gap is filled by the same recombinational process just described for postreplication repair Recombination repair Damaged DNA was not removed,but after new

19、 DNA was made and cell division,the damage DNA was cut off.A model for non-homologous end joining (NHEJ). DNA ends are first bound by the Ku80/70 7heterodimer, which then attracts DNA-PKcs to form the DNA-PK complex. DNA-PK attracts Artemis, APTX, PNKP, polymerase and polymerase , as well as ligase IV complex (comprised of ligase IV, XRCC4 and XLF), which together seal the DNA endsAn assumed mechanism for the repair of DNA double-strand breaks by homologous recombination (HR). HR utilizes a homologous stretch on a sister chromatid to accurately repair the DSBSOS system SOS repair is for emer