The Ames test indicates the mutagenic potential of a compound

Transcription

1 The Ames test indicates the mutagenic potential of a compound Developed by Bruce Ames Uses Salmonella strain with a mutation that makes bacterium unable to synthesize His Add compound to plate of Salmonella, see if it grows in His free medium Colonies (+ test) indicates the compound mutated the Salmonella, restored ability to synthesize His

2 The Ames Test

3 2) DNA repair system 의유형 * DNA repair DNA 의 2 개의상보적가닥중한쪽가닥 DNA 에손상 ; 제거 손상받지않은 DNA 가닥 ; 주형으로하여치환 1) Mismatch repair: MuS, MutL, MutH, exonuclease, DNA polymerase III, ligase 2) Base-excision repair 3) Nucleotide-excision repair 4) Direct repair 5) recombinational repair * Repair mechanism ⅰ) 손상小 : 이상염기부분을 DNA glycosylase로제거 Exonuclase phosphodiester 결합절단 5 3 exonuclease가 5 쪽 nucleotide 제거 DNA polymeraseⅠ, DNA ligase에의해수복 ⅱ) 손상大 : Endonuclease가손상양끝의 phosphodiester 결합절단, 제거 DNA polymeraseⅠ 이바른염기배열형성되게함 DNA ligase가연결

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5 Types of DNA Damage Mismatches arise from occasional incorporation of incorrect nucleotides Abnormal bases arise from spontaneous deamination, chemical alkylation or exposure to free radicals Pyrimidine dimers form when DNA is exposed to UV light Backbone lesions occur from exposure to ionizing radiation, free radicals

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7 Methylation of DNA and Repair The newly synthesized strand is unmethylated for a short period after synthesis Any replication errors must reside in the unmethylated strand Methyl directed mismatch repair system will cleave the unmethylated strand in the initial part of the repair process

8 (1)Mismatch Repair and Methylation

9 Methylation directed Mismatch Repair in E. coli: Early Steps

10 Methylation directed DNA Repair in E. coli: Later Steps

11 (2) Base excision repair uses specific DNA glycosylases DNA glycosylases recognize specific lesions cleave N glycosyl bond between sugar and base Creates apurinic/apyrimidinic (AP) site Examples of DNA Glycosylases Uracil glycosylase removes uracil from DNA Important because C spontaneously deaminates to U; U doesn t belong in DNA Deamination is 100 faster in ssdna Other glycosylases make AP sites at 8 hydroxyg, hypoxanthine, 3 methyladenine, etc.

12 Repair at AP Sites in Bacteria The entire nucleotide is ultimately removed, not just the damaged base In fact, sometimes the region around the AP site is removed AP endonucleases cut the DNA backbone around the AP site, remove DNA DNA Pol I synthesizes new DNA DNA ligase seals the nick

13 (2)Base excision repair

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15 (3)Large distortions in DNA are repaired by nucleotide excision Lesions include: pyrimidine dimers (formed from UV light) 6,4 photoproducts (from UV light) benzo[a]pyreneguanine (from cigarette smoke) Pathway involves removal of a DNA segment by excinucleases

16 Nucleotide excision repair in bacteria uses the ABC excinuclease Excinucleases cleave DNA backbone in two places ABC excinuclease contains UvrA, UvrB, and UvrC subunits hydrolyze fifth bond on 3 side of the lesion and eighth bond on 5 side Remove nucleotides DNA Pol I and DNA ligase to replace the DNA and seal the gap

17 (3)Nucleotide Excision Repair in Eukaryotes Hydrolyze sixth bond on 3 side and twenty second bond on 5 side of lesion Remove nucleotides In humans, gap filled using DNA polymerase and DNA ligase

18 NER in Bacteria and Humans Xeroderma pigmentosum: excinuclease 결손

19 (4)Direct Repair Photolyases use light energy to repair pyrimidine dimers Enzymes not found in humans and placental mammals O 6 methylguanine DNA methyltransferase repairs methylated guanine AlkB demethylates 1 methyladenine and 3 methylcytosine

20 Repair of Pyrimidine Dimers with Photolyase N 5,N 10 -methenyltetrahydrofolylpolyglutamate (MTHFpolyGlu)

21 How O 6 Methylguanine Leads to Mutation O 6 methylguanine DNA methyltransferase

22 Direct Repair of Alkylated Bases by AlkB

23 Carcinogenesis process

24 DNA Recombination Segments of DNA can rearrange their location within a chromosome from one chromosome to another Such recombination is involved in many biological processes Repair of DNA Segregation of chromosomes during meiosis Enhancement of genetic diversity In sexually reproducing organisms, recombination and mutations are two driving forces of evolution Recombination of co infecting viral genomes may enhance virulence and provide resistance to antivirals

25 기능 : DNA repair, 염색체의상호작용, 유전적다양성 FIGURE 25-32a Crossing over.

26 (5)Recombinational DNA Repair Breast cancer: BRCA1,2 결함 ( 여성 80%); recombinational repair 저하

27 6) Nonhomologous End joining (NHEJ) :Ku70-Ku80 complex 가 DNA 말단결합 그림 25-37

28 Genomics has led us to a better understanding of the molecular signals of the disease Genome How Does Personalized Medicine Come Real Clinical Data Metabolome Health Information Proteome

29 0.5 % difference What We Know? Genetic Variant

30 The Future of Medicine Primary Care Newborn screening expands to track hundreds or thousands of genes, rather than dozens. Information is used throughout the Blood and other individual s life to samples drawn during manage their routine medical visits healthcare. are submitted for molecular screening for a large number of cardiovascular, cancer, l i l d th Medical information on a smartcard contains our unique molecular profile. Healthcare providers will

31 4) Genetic recombination 1 homologous genetic recombination 2 site-specific recombination 3DNA transposition DNA repair, 유전자발현, 염색체분리, 유전적다양성등 5) Transposon: DNA transposition=jumping gene 6) Ig recombination

32 Transposable Genetic Elements (Transposons) Certain DNA segments can change their relative position (move to a region or a new chromosome) Carry genes for transposases, but some can contain extra genes (e.g., antibiotic resistance) In bacteria, short (5 10 bp) sequences act as binding sites for transposases These sites become duplicated See Fig

33 Insertion of a Transposon

34 Immunoglobulin genes assemble by recombination Although human genome has <30,000 genes, millions of antibodies (immunoglobulins) can be created

35 Kappa chain V, J, and C segments undergo recombination Kappa chain gene has ~300 V (variable segments) ~4 J (joining) segments 1 C (constant) segment = 1200 possible V J arrangements V J junction sequence variation: to 3000 possible combinations Heavy chain and lambda chains have more combinations: 5,000 Ultimately > possible IgG molecules! 3,000X5,000

36 Recombination of the V and J Gene Segments of the Human IgG Kappa Light Chain in B cell = 1200 possible V J arrangements

37 < 요약 >

38 DNA Replication in E. coli Requires a Family of Proteins

39 TABLE 25-5 Types of DNA Repair Systems in E. coli

40 Genetic recombination 1 homologous genetic recombination 2 site-specific recombination 3DNA transposition - DNA repair, 유전자발현, 염색체분리, 유전적다양성등 Transposon: DNA transposition = jumping gene Antibody diversity DNA damage and cancer