Chapter 16. Mechanisms of Genetic Variations. Laboratory of Bioantibiotics College of Pharmacy, CHA University

Transcription

1 Chapter 16. Mechanisms of Genetic Variations 1 미생물의유전 미생물유전자의변이및전달

2 2 공부할내용 유전자변이 (mutations) mutation 의종류 : point mutations (substitution/indel) mutation의영향및결과 :??, Ames test ( 발암원검증 ) 유전자수복 (DNA repair) systems that safeguard DNA integrity (sequence, structure) proofreading vs DNA repair directrepair, repair, excisionrepair (BER, NER) mismatch repair, recombination repair, error prone repair (SOS response) 유전자전달 (gene transfer) vertical gene transfer horizontal gene transfer (HGT): en bloc gene transfer 세균의유전물질 : transposable elements, plasmids, bacteriophages conjugation: F plasmid. Hfr cell. F plamid transformation: competence and DNA uptake transduction: generalized vs specialized transduction

3 3 Mutations stable, heritable changes in sequence of bases in DNA point mutations most common: substitution, insertion, deletion alteration of single pairs of nucleotide (transition and transversion) addition or deletion of nucleotide pairs (frameshift in a coding region) larger mutations are less common insertions, deletions, inversions, duplication, and translocations of nucleotide sequences mutations can be spontaneous or induced 돌연변이

4 4 Spontaneous mutations arise without exposure to external agents may result from errors in DNA replication due to base tautomerization resulting in transition and transversion mutations due to insertion or deletion of nucleotides may also result from the action of mobile genetic elements such as transposons 돌연변이

5 5 Spontaneous mutations Non-Watson-Crick base paring due to tautomerism 돌연변이

6 6 Spontaneous mutations Insertions and deletions (Indels Indels) 돌연변이

7 7 Induced mutations caused by agents that directly damage DNA base analogs structurally similar to normal bases mistakes occur when they are incorporated into growing polynucleotide chain DNA modifying agents alter a base causing it to mispair intercalating agents distort DNA to induce single nucleotide pair insertions and deletions 돌연변이

8 8 Induced mutations MNNG(=NTG) and thymine dimer 돌연변이

9 9 Induced mutations Nitrous acid (free acid at ph = 4.25) reacts with amino groups to convert them into hydroxyls (i.e. deamination) cytosine uracil adenine hypoxanthine guanine xanthine other parts of DNA/RNA is not very susceptible to deamination nitrous acid is used as a mutagenizing reagent (transition) 돌연변이

10 10 Induced mutations Hydroxylamine (NH 2 OH) reacts with the NH 2 of cytosine (more slowly with NH 2 of adenine) : NH 2 NHOH (hydroxyamino) favoring tautomerization increasing transition frequency (CG to TA) during DNAreplication in vitro. similar to methoxyamine (NH 2 OCH 3 ) cf. hydroxylamine y cleavage of protein toxic effects in vivo. 돌연변이

11 11 Induced mutations EMS mutagenesis cf. 8-oxoG 돌연변이

12 12 Induced mutations 5-BU (bromouracil) cf. 2-AP (aminopurine) 돌연변이

13 13 Effect of mutations wild type most prevalent form of gene forward mutation wild type mutant form reversion mutation mutant phenotype wild type phenotype suppressor mutation occurs when the second mutation is at a different site than the original mutation 돌연변이의결과

14 14 Types of point mutations Substitution transition, transversion Frameshift Indel of a base pair Silent (= synonymous) y Missense Nonsense Frameshift 돌연변이의결과

15 15 Mutations in coding regions point mutations in protein coding genes can affect protein structure in a variety of ways are named according to if and how they change the encoded protein the most common types are: silent, missense, nonsense, and frameshift mutations silent mutation change nucleoside sequence of codon but not the encoded amino acid missense mutation a single base substitution that changes codon for one amino acid into codon for another amino acid nonsense mutation converts a sense codon to a stop codon frameshift mutation tti results from insertion or dlti deletion of one or two base pairs in the coding region of the gene 돌연변이의결과

16 16 Mutations at work conditional mutations expressed only under certain environmental conditions auxotrophic mutant unable to make an essential macromolecule such an amino acid or nucleotide has a conditional phenotype wild type strain from which it arose is called a prototroph mutations in regulatory sequences conditional lac operon mutants many of these mutations map in the operator site and produce altered operator sequences not recognized by repressor operon is always transcribed and b galactosidase is always synthesized mutations in trna and rrna genes protein synthesis is disrupted 돌연변이의결과

17 17 Detection and isolation of mutants mutations are generally rare one per 10 7 to cells finding mutants requires sensitive detection methods and/or methods to increase frequency of mutations observation of changes in phenotype replica plating technique used to detect auxotrophic mutants use of environmental condition in which h only desired dmutant twill grow eg e.g., selection for revertants from auxotrophy to prototrophy 돌연변이실험

18 18 Detection and isolation of mutants Replica plating 돌연변이실험

19 19 Carcinogenicity testing based on observation that most carcinogens are also mutagens tests for mutagenicity are used as screen for carcinogenic potential e.g. Ames test reversion rate in presence ofsuspected carcinogen > reversion rate in absence of suspected carcinogen then, agent is a mutagen, and may be carcinogen 돌연변이실험

20 20 Safe-guarding systems Systems that safeguards DNA Restriction/modification system: protecting from foreign DNA Repair system: protecting from damages Hd Hsd system: confers self specificity it (methylating ti A) Dam system: distinguishes the new DNA (methylating A) Dcm system: unknown function (methylating C) Dealing with injuries in DNA any change introducing structural deviation by single base changes (overall structure is usu. unchaged) structural distortions: physical impediment to replication or transcription (e.g. single strand nick, removal of a base, pyrimidine dimer etc) DNA 수복

21 21 DNA repair systems Proofreading by DNA polymerase correction of errors in base pairing made during replication errors corrected by DNA polymerase Repair by DNA repair systems that also work specific DNA repairs using N glycosylases: AP sites generated other specific repairs: reversal of the DNA damages (= direct repair) mismatch repair: scrutinizing DNA to minimize misincorporation excision repair: removal of the DNA damage and re synthesis to replace the excision (NER and BER) repairs working during replication (= DNA damage tolearance pathways) recombination repair: recombination with another intact copy translesion synthesis (= error prone repair): the SOS response/repair DNA 수복

22 22 Specific repair pathways DNA repair systems recognize the altered bases N glycosylases remove altered bases, resulting in AP sites AP endonucleases cut the 5 P of the AP nucleotides DNA PolI re synthesizes DNA from the 3 OH (nick ktranslation) ti Examples of altered bases recognized Deamination: G, A, T/U ROS mediated oxidation: 8 oxog Alkylation: G O6, T O4 > G N7, A N3, P UV irradiation: thymidine dimers (cyclobutane dimer, 6 4 dimer) DNA 수복

23 23 Specific repair of 8-oxoG 8 oxog 제거 A 제거 DNA 수복 N glycosylases: MutM, MutY 8 oxogtp phosphatase: MutT

24 M/DEET 문항 돌연변이 (3 차시험문제예고 )

25 25 Direct repair pathways Not mediated by N glycosylases, but just reversed by specific enzymes direct repair of alkylated bases catalyzed by alkyltransferase or methylguanine methyltransferase direct repair of UV damaged bases (= photoreactivation) used to directly repair thymine dimers thymines separated by photochemical reaction catalyzed by photolyase DNA 수복

26 26 General repair pathways DNA repair systems recognize the structural distortion not modification specific Examples of distorted structures recognized Base analogs (2 AP, 5 BU) Frameshiftmutagens (= intercalating i agents) Examplesofgeneral repair pathways Mismatch repair Excision repair DNA damage tolerance pathways: recombination repair/sos repair DNA 수복

27 27 Mismatch repair type of excision repair e.g., mismatch repair system in E. coli mismatch correction enzyme scans newly synthesized DNA for mismatched pairs mismatched pairs removed and replaced by DNA polymerase and DNA ligase needs to know which strand is correct DNA 수복

28 28 DNA methylation used by E. coli mismatch repair system to distinguish old DNA strands from new DNA strands hemi methyalted: old DNA (template t strand) methylated; new DNA unmethylated the repair system cuts out the mismatch from the unmethylated strand catalyzed by DNA methyltransferases Methylation-directed mismatch repair DNA 수복

29 29 Excision repair corrects damage that causes distortions in double helix two types of repair systems are known nucleotide excision repair (NER): nt baseexcisionrepair excision (BER) both remove the damaged portion of the DNA strand and use the intact complementary strand as a template to synthesize new DNA DNA 수복

30 30 Excision repair DNA 수복

31 31 Recombination repair repairs DNA with damage in both strands involves recombination with an undamaged molecule in rapidly dividing cells, another copy of chromosome is often available RecA protein catalyzes recombination events DNA 수복

32 32 Translesion synthesis (TLS) The SOS response as an inducible repair system a global control network used to repair excessive damage that halts replication, leaving many gaps when repressor LexA is destroyed, numerous genes that are negatively regulated are activated RecAprotein initiates recombination repair RecAprotein also acts as coprotease, enhancing the cryptic protease activityoflexa repressor protein and thereby increasing production of repair enzymes DNA polymerases IV and V synthesizes unrepaired DNA PolIV (=DinB) : DinB interacts with UmuD 2 and RecA PolV mutasome: UmuD 2 C RecA ATP DNA 수복

33 33 Creating genetic variability veritical gene transfer followed by mutations and recombinations mutations are subject to selective pressure each mutant form that survives becomes an allele, an alternate version of a gene recombination is the process in which one or more nucleic acids are rearranged or combined to produce a new nucleotide sequence (recombinants) 유전자전달

34 34 Horizontal gene transfer (HGT) HGT differs from vertical gene transfer transfer of genes from one independent, mature organism to another HGT of replicable genetic elements (e.g. conjugable plasmids or phages) )facilitates autonomous replication of the elements HGT of non replicacble gene blocks leads to the formation of genomic islands (i.e. en bloc transfer), which h are important t in evolution of many species expansionofecologicalniche of ecological niche, increasedvirulence (e.g. pathogeneicity islands) 유전자전달

35 35 HGT mechanism occurs in the three mechanisms evolved by bacteria to create recombinants genes can be transferred to the same or different species transfer of gene from donor to recipient exogenote DNA that is transferred to recipient endogenote genome of recipient merozygote recipient cell that is partial diploid as result of transfer process 유전자전달

36 36 HGT mechanism Exogenote DNA 의운명 유전자전달

37 37 Recombination following HGT three types homologous recombination: RecA mediated site specific recombination transposition: transpoase (encoded dby the exogenote) Bacterialgenetic elements in regards to HGT free DNA from ruptured cells transposable elements plasmids (see Chapter 3) focusing on F plasmid and conjugation bacteriophages 유전자전달

38 38 Homologous recombination most common usually involves a reciprocal exchange between pair of DNA molecules with same nucleotide sequence RecA proteins carry out double strand break most accepted 유전자전달

39 39 Homologous recombination Nonreciprocal homologous recombination incorporation of single strand of DNA into chromosome, forming a stretch of heteroduplex DNA proposed to occur during bacterial transformation 유전자전달

40 40 Site-specific recombination important in insertion of viral/ genome into host chromosomes there is only a small region of homology between inserted genetic material and host chromosome 유전자전달

41 41 Transposable elements segments of DNA that move about the genome in a process called transposition simple transposition vs replicative transposition are sometimescalled jumpinggenes genes the simplest transposable elements are IS elements transposable elements which containgenes other than those used for transposition are called transposons 유전자전달

42 42 Transposons simple transposition replicative transposition 유전자전달

43 43 Transposons Size (bp) Target DNA size Functions /Terminal lirs IS (20/23) IS2 1,327 5 (32/41) IS10 1,329 9 (17/22) IS21 2,132 4 (10/11) Tn3 4,957 5 (38/38) Ap resistance Tn5 5,700 9 (IS50 8/9) Km Sm Bm resistance Tn10 9,300 9 (IS10 17/22) Tc resistance Tn3 in R plasmid 유전자전달

44 44 Plasmids autonomously replicating DNA molecules that can exist independently d (i.e. as episomes), integrate t reversibly into the host chromosome see Chapter 3 유전자전달

45 45 Plasmids autonomously replicating DNA molecules that can exist independently d (i.e. as episomes), integrate t reversibly into the host chromosome conjugative plasmids such as the F plasmid can transfer copies of themselves to other bacteria during conjugation 유전자전달

46 46 F plasmid F factors contain the information for formation of sex pilus attach F + cell to F cell for DNA transfer during bacterial conjugation F factors have insertion sequences (IS) assists in plasmid integration Hfr cells 유전자전달

47 47 Bacterial conjugation J. Lederberg and E. Tatum demonstrated the transfer of genes between bacteria that depends on direct cell to cell contact mediated by the F pilus (sex pilus) unidirectional DNA transfer from donor to recipient i cf. Conjugation is the transfer of the mobilizable (Mob + ) DNA using the mobilizing/transferring (Tra + ) function of the donor cell : the Mob function include ( ) : the Tra function include ( ) 유전자전달

48 48 F + x F - mating a copy of the F factor is transferred (from orit) to the recipient i and does not integrate into the host chromosome F plasmid encodes sex pilus a Type IV secretion system that makes contact tbt between cells that t DNA moves across Retraction occurs once the contact is made F plasmid is replicated by rolling circle method (= σ mode) 유전자전달

49 49 Sex pilus & σ replication (orit) 유전자전달

50 50 Hfr conjugation donor Hfr cell has F factor integrated into its chromosome donor genes are transferred to recipient cell a complete copy of the F factor is usually not transferred gene transfer can be clockwise or counterclockwise 유전자전달

51 51 Hfr conjugation 유전자전달

52 52 F' conjugation result when the F factor incorrectly leaves the host chromosome some of the F factor is left behind in the host chromosome some host genes have been removed along with some of the F factor these genes can be transferred to a second host cell by conjugation 유전자전달

53 53 Transformation F. Griffith demonstrated transformation uptake of naked DNA by a competent cell followed by incorporation of the DNA into the recipient cell s genome 유전자전달

54 54 Competence and uptake systems protein system allows DNA to move across gram positive negative cell walls PilQ aids in movement across outer membrane Pilin complex (PilE) movesdnaacross periplasm and peptidoglycan ComEis DNA binding protein N is nuclease that degrades one strand ComA forms transmembrane channel Neisseria gonorrhoeae Bacillus subtilis 유전자전달

55 55 Transduction N. Zinder and J. Lederberg discovered the virus mediated bacterial gene transfer viruses can carry out the lytic cycle in which the host cell is destroyed dor the viral DNA can integrate into the host genome, becoming a latent prophage 유전자전달

56 56 Generalized vs specialized Generalized trasduction by generalized transducing particles any part of bacterial genome can be transferred occurs during lytic cycle of virulent phage during viral assembly, fragments of host DNA mistakenly packaged into phage particles Specialized trasduction by specialized transducing particles specific part of bacterialgenomeistransferred is transferred carried out only by temperate phages that have established lysogeny occurs when the prophage is incorrectly excised 유전자전달