Chap_25_2010_v2 - Chapter 25 DNA metabolism> Faithful...

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Unformatted text preview: Chapter 25: DNA metabolism -> Faithful copies (Lecture 5) DNA replication and DNA polymerases (Lecture 6) The different replication steps -> Corrections of errors (Lecture 6-7) Mutagenesis and DNA repair -> Introduction of genetic variability (evolution) (Lectures 7) Homologous DNA recombination & Site-specific DNA recombination (Lecture 6) -- DNA replication and DNA polymerases -- The different replication steps --- Initiation --- Elongation --- termination 1 Enzymes involved in the DNA metabolism Case study : DNA replication in E. Coli (prokaryotes) Terminology (e.g) 1 min = ~40000 bp Genes are italicized dnaA, replication PolC, polymerase uvrB, UV resistance recA, recombination (Letters reflect order of discovery) Enzymes (genes products) DnaA protein PolC = DNA polymerase III RecA protein Map of E. Coli chromosome with genes involved in DNA metabolism Replication is a direct consequence of the DNA structure Strict base pairing rules imply that each strand provides the template for a sister strand with a predictable and complementary sequence Double helix 3’ 5’ Base pairin Base Ribose phospha backbo Fundamental properties and mechanisms of replication are universal among living organisms -> Replication Fundamental Rules 5’ 5’ New strands 3’ 3’ 2 Different possible models for replication Replication is semi-conservative (suggested by Watson & Crick) The Meselson-Stahl experiment (1957) E. Coli cells culture is performed in presence of NH4Cl (15N). The density of 15N DNA is 1% greater than 14N DNA and can thus be separated on cesium chloride density gradient. The cells culture is transferred to a fresh medium containing only 14N and allowed to grow until the population has doubled (b) and quadrupled (c). Replication is semi-conservative 3 (1) 3H thymine labeling of replicating DNA visualized by autoradiography (John Cairns) -> Track of silver grains corresponding to 3H DNA (2) Electron micrographs Replication begins at an origin! Is replication uni-directional or bi-directional? Experiment with 3H thymine mixed with dividing E. coli cells for a short period of time Radioactive 3H DNA -> Replication is bi-directional Replication loops originate generally at a unique point in the DNA (prok). This point is called an origin (ori). (Demonstration by denaturation mapping (denatured A-T bubbles served as landmarks to identify ori)) 4 Rule: DNA synthesis always proceeds in the 5’ to 3’ direction and is semi-discontinuous (template is read from 3’ to 5’) Defining DNA strands at the replication fork Continuous synthesis in the same direction as replication fork movement Few 100 to few 1000 bp Discontinuous synthesis in the direction opposite to the fork movement DNA is synthesized by DNA polymerases At least 5 different polymerases in E. Coli. First discovered = DNA polymerase I (Kornberg, 1955) All DNA polymerases catalyse a common, fundamental phosphoryl group transfer reaction : (dNMP)n + dNTP -> (dNMP)n+1 + ppi Lenghtened DNA 5 Degraded by pyrophosphatase Requirements for DNA polymerization: (1) template, (2) primer (3’ end is called primer terminus, primers are often RNA rather than DNA. The average number of nucleotides added before a polymerase dissociates defines it processivity. 2 to 3 conserved Asp that chelate two magnesiums involved in catalysis DNA polymerases are metallo-enzymes! 6 Replication is very accurate High degree of fidelity Polymerases exclude incorrectly paired bases In E. coli One mistake every 109 to 1010 nt added. One error per 1000 to 10000 replications. Discrimination between correct and incorrect nt relies on recognition of proper pairing and geometry. Polymerase activity fidelity of 104 to 105. AT and CG bp are isosteric DNA is degraded by nucleases or Dnases (Reminder from Chapter 9). Exonucleases: degrade nucleic acids from one end. Some are specific for removing nt from the 5’ or 3’ end of one strand of a double stranded DNA, operating in the 5’ to 3’ direction, or 3’ to 5’ direction respectively. Endonucleases: degrade DNA at any site, reducing it to small fragments. Restriction endonucleases: cleave DNA only at specific nucleotide sequences (Chapter 9). (Some exo- and endo-nucleases degrade only single stranded DNA.) EcoR1 restriction enzyme David S. Goodsell 7 E. coli has at least 5 DNA polymerases DNA pol I (1955, Kornberg). DNA Pol II involved in DNA repair (1970): as well as DNA Pol IV and V (1999) DNA Pol III is the principal replication enzyme in E. coli (1970). DNA Polymerase Proofreading Activity 3’-> 5’ exonuclease activity increases replication fidelity by 102 to 103 fold. This activity is called proofreading. 8 DNA Polymerase Proofreading Activity 3’-> 5’ exonuclease activity increases replication fidelity by 102 to 103 fold. This activity is called proofreading. Incorporation of an A-C mispairing Blockage of the elongation Polymerase slides back to remove the mispaired nt by 3’->5’ exonuclease activity Elongation is resumed One net error for every 106 to 108 base added. Additional accuracy provided by DNA repair DNA Polymerase I DNA pol I performs a host of cleanup function during replication, recombination and repair. Klenow fragment of E. coli Pol I responsible for Pol activity and proofreading activity. Nick translation activity The intact DNA pol I has also a 5’->3’ exonuclease activity that can replace a segment of RNA or DNA paired to the template. 9 DNA Polymerase I 5’->3’ polymerase activity DNA 3’->5’ exonuclease activity (Proofreading) 5’->3’ exonuclease activity (Nick translation) Taq DNA polymerase (Chap. 9) David S. Goodsell E. coli has at least 5 DNA polymerases DNA pol I (1955, Kornberg). DNA Pol II involved in DNA repair (1970): as well as DNA Pol IV and V (1999) DNA Pol III is the principal replication enzyme in E. coli (1970). 10 DNA pol III β clamp End view Side view The two β subunits of E. coli Pol III form a donut-shaped circular clamp that surrounds DNA. The β clamp prevents the dissociation of DNA from DNA Pol III and increase processivity to greater than 500000 nts 11 DNA replication requires many enzymes and protein factors. DNA Replicase System or Replisome involves at least 20 different enzymes (in E. coli) that work in a highly coordinated way. It is also highly regulated. Enzyme DNA Polymerases Helicases Topoisomerases DNA binding proteins Primases DNA ligases Function Synthesize DNA strands Separate DNA strands (require ATP) Relieve DNA topological stress Stabilize separated strands Synthesize RNA primers (note: primers are removed by DNA Pol I) Seal DNA nicks The different stages of DNA replication (info from in vitro experiments with purified E. coli proteins) Initiation, elongation, termination 12 The Initiation Step Replication origin oriC: 245bp sequence: conserved among bacterial replication ori. At least 9 ≠ proteins participate to the initiation phase. Model for initiation of replication at oriC (AAA+ ATPases) (1) Complex of ~8 DnaA proteins binds at the R and I sites in the origin. It forms a righthanded helical structure that lead to the denaturation of the AT rich DUE region (ATP dependent). This process is facilitated by HU, IHF and FIS. (2) Hexamers of the DnaB protein bind to each unwound strand with the help of DnaC. (3) The DnaB helicase activity further unwind the DNA for creating two potential replication forks. 13 Timing for DNA replication is affected by methylation and binding to bacterial plasma membrane OriC DNA region is highly enriched in GATC palindromic sequences which are methylated by Dam methylase. 11/256bp (ori) versus 1/256 (44) (random). After methylation the oriC DNA is hemi-methylated and sequestered for a period by the plasma membrane. oriC must be released and methylated by Dam methylase before it can be bound to DnaA and initiate replication. The Elongation Step Relieves DNA topological stress Synthesizes elongating DNA strand Separates DNA strands (require ATP) { = Dna G Primosome Synthesizes 10-60nt RNA primers Stabilizes separated strands Synthesis of leading and lagging strand are different. They are running in the opposite direction! Solution! 14 The Choreography of DNA Replication Both strands are produced by a single assymetric DNA Pol III dimer 15 16 17 This process allows rapid synthesis of DNA (1000nt/second) Once the synthesis of an Okazaki fragment is completed: 1) DNA by Pol I replaces RNA primer by DNA 2) DNA ligase links two adjacent Okazaki fragments 18 DNA ligase DNA ligase requires ATP (Eukaryotes, viruses) or NAD+ (bacteria) (1) Chap 14! (3) (2) 19 Summary: proteins at the E. coli replication fork The Termination Step Few 100 bp Terminus region opposite to ori has multiple copies of 20 bp sequence called, Ter (terminus). Tus protein binds to Ter. Tus-Ter complexes act as a trap for the replication fork (arrest) replication from one direction. Mechanism unknown “Catenanes” DNA topoisomerase IV (type II) 20 21 Replication in Eukaryotes It is more complex butit has essential features similar to prokaryotes and viruses Multiple origins of replication (replicators or ARS (Autonomously Replicating Sequences) ~ 400 ARS in genome of yeast (150 bp long) ORC multisubunit protein is required for initiation (Origin Recognition Complex) -> binding to replicators is regulated Inititation of replication at eukaryote replication origin Pre-replicative complex (pre-RC) 22 DNA replication in Eukaryotes Rate of replication: 50 nt / second Start from multiple origins spaced by 30000 to 300000 bp Several DNA polymerases for nuclear genomes DNA Pol α -- Multisubunit enzyme (similar in all Euk. Cells) -- Primase activity, polymerase activity -- No 3’->5’ proofreading activity => Responsible for the synthesis of primers for Okazaki fragments DNA Pol δ -- Extend primers --Associate with PCNA (proliferating Cell Nuclear Antigen) <=> to β subunit of DNA Pol III => Equivalent to DNA Pol III! DNA Pol ε -- involved in DNA repair => similar to DNA Pol I Other proteins RPA <=> SSB RFC is a clamp loader (or match maker) for PCNA and facilitate replication complexes Termination of replication of linear chromosomes involves the synthesis of telomeres (Lecture 7) -- Part 2: Mutagenesis and DNA repair -- Part 3: DNA recombination --- Homologous genetic recombination --- Site-specific recombination --- DNA transposition 23 Part 2: Mutagenesis and DNA repair DNA repair is essential for preventing DNA damage in cells Definition: mutation Permanent change in the nucleotide sequence is a mutation. Some mutations can have deleterious effect leading to cancer. (Box 25-1) A silent mutation is a mutation that affects nonessential DNA or with negligible effect. Without DNA repair, damaging reactions would make life impossible (>1000 lesions in mammalian cell/24h that need repair). Illustration: Ames test (Fig. 25-21): A measure of the mutagenic power of a chemical (mutagenic power is correlated to its carcinogenic power) Strain of bacteria with a mutation that inactivates an enzyme in the His biosynthetic pathway. If compound is mutagenic, it will increase the rate of back-mutation and hence the number of colonies. His free medium Mutagen [mutagen] (b) (c) (d) 24 Different types of mutations in DNA 25 Spontaneous Depurination Deamination Chapter 8 p.290-291 Alkylation Photo products 26 All cells have multiple DNA repair systems (A)-> If lesions in double-stranded DNA with one correct strand (1) (2) (3) (4) Mismatch repair Base-excision repair Nucleotide-excision repair Direct repair (B) -> If double strand break lesions or lesions in single strand DNA: when a replication fork encounter a DNA damage Last resort repair solution --Recombinational DNA repair --Error-prone repair -> SOS response Repair is very energy consuming! When the genetic info is in danger, the amount of chemical energy invested to repair is almost irrelevant! Repair is possible because DNA is double stranded! (1) Mismatch repair (after replication) Correction of mismatches just after replication improves fidelity by 100 to 1000! “Methyl-directed mismatch repair” -> Methylation of the DNA strands allows to discriminate between the template and the newly synthesized strand. Just after replication the DNA is hemimethylated (the new strand has no methyl on A (N6)) -> Replication mismatches at the vicinity of a hemimethylated GATC sequence can be corrected according to the methylated parental strand. -> If both strands are methylated few mismatches are repaired (in vitro experiments) Importance of good timing! 27 Methylation of the DNA strands allows to discriminate between the template and the newly synthesized strand. Just after replication the DNA is hemimethylated (the new strand has no methyl on A (N6)) 28 -> Replication mismatches at the vicinity of a hemi-methylated GATC sequence can be corrected according to the methylated parental strand. -> If both strands are methylated few mismatches are repaired (in vitro experiments) Good timing is important! Methyl-directed mismatch repair The 12 players Dam methylase MutH, MutL, MutS DNA helicase II SSB DNA polymerase III Exonuclease I Exonuclease VII Exonuclease X RecJ nuclease DNA ligase MutL-MutS complex binds to all mismatches (except C-C). 29 MutL-MutS complex binds to all mismatches (except C-C). Mismatch MutH binds to MutL and Methyl-GATC. DNA is threaded on both side of the mismatch, creating a loop. 30 MutH is a specific endonuclease that cleaves only when the MutLMutS encounter a Met-GATC (either on the right or left side of the mismatch): it cleaves the unmethylated strand on the 5’ of the G. Methyl-directed mismatch repair Pathway of repair depends on the location of the cleavage site relative to the mismatch. 3’ side 5’ side (5’->3’ or 3’->5’) 5’->3’ 3’->5’ Expensive: mismatch can be at several 1000bp from GATC. (Estimate the cost for the cell) (For eukaryotes, the system of mismatch repair remains partially unknown) 31 (2) Deamination, depurination and are taken care by the base-excision repair pathway Depurination of DNA is promoted under normal cellular conditions (1/105/cell/24h) Deamination is promoted by nitrous acid (HNO2) and nitrites (e.g.NaNO2) Why DNA has T and not U? The fact that DNA has thymine instead uracil allows to distinguish selectively thymine from uracil resulting from deamination of cytosine. (2) Base-excision repair Repair damages that do not lead to major changes in the structure of the DNA backbone d Abasic or AP site (1) DNA Glycosylases remove affected bases by cleaving the Nglycosidic bond. -> Specific DNA glycosylases for one type of lesions. Example: Uracil glycosylase Once an AP site is formed: (2) An AP endonuclease cleaves the backbone near the AP site. (3) DNA polymerase I initiates repair synthesis from the free 3’ OH at the nick, removing a portion of the damage strand (with its 5’->3’ exonuclease activity) and replacing it with undamaged DNA (4) The remaining nick is sealed with DNA ligase 32 Mutations resulting from radiation Taken care off by: (3) Nucleotide excision repair (4) direct repair pathways Photo product dimers induce a distortion of the DNA helical structure This type of lesions can be repaired by the nucleotide-excision repair system. Backbone kink 33 (3) Nucleotide-excision repair Excinucleases: multisubunit enzymes UvrABC Excinuclease UvrC 8th bond UvrB 5th bond 22nd bond (16 polypeptides) 6th bond UvrD In E. coli: ABC excinuclease (UvrA, UvrB, UvrC) A2B binds to the site of the lesion. UvrA dissociates, leaving a tight Uvr B -DNA complex. UvrC binds to UvrB and both UvrB and UvrC cleave the DNA on both side of the lesion. UvrD (helicase) removes the DNA fragment. In human: deficiencies in this pathway result to cancer (eg: XP Xeroderma Pigmentosum) (4) DNA Direct repair (a) repair of thymine dimers 34 (a) DNA Direct repair UV radiation damages Direct photoreactivation of cyclobutane pyrimidine dimers -> catalyzed by photolyases. -> Photolyases use energy from absorbed light to reverse the damage. (MTHF) Methenyltetrahydrofolylpolyglutamate <=> photo-antenna (b) DNA direct repair of alkylated bases Alkylating agents can alter certain bases of DNA. By methylating guanine -> O6 methyl guanine that can only pair to thymine! DNA lesion 35 DNA Direct repair of O6 methylguanine (should occur before replication) The solution for repair How DNA damage can result in mutation! This reaction is performed by O6 methylguanineDNA methyltransferase. Single turnover enzyme! Inactivated after methyl transfer! Inactivated protein functions as a transcriptional activator. (increase expression of its own gene and the genes for few other repair enzymes.) Another direct repair mechanism: Direct repair of alkylated bases by AlkB Oxidative demethylation 36 (B) DNA damage and its effect on DNA replication Can lead to error-prone translesion DNA synthesis Part 3: DNA Recombination (A) Homologous genetic recombination Involves genetic exchanges between pieces of the chromosome that share an extended region of nearly identical sequence. Occurs in all cells, during meiosis and mitosis in eukaryotes. (B) Site-specific recombination The DNA exchanges occur only at a particular DNA sequence. (C) DNA transposition Involves a short segment of DNA with the capacity to move from one location in a chromosome to another (first discovered in maize by Barbara McClintock, 1940s). 37 Introduction Why DNA recombination is desirable? (a) Specialized DNA repair systems (b) Specialized activity in DNA replication (c) Regulation of expression of certain genes (d) Facilitation of proper chromosome segregation during cell division in eukaryotes (e) Maintenance of genetic diversity (-> promote evolution) (f) Implementation of programmed genetic rearrangements during embryonic development The three main functions of homologous recombination (1) provides, in eukaryotic cells, a transient physical link between chromatids that promotes the orderly segregation of chromosomes during mitosis and at the first meiotic cell division (2) enhances the genetic diversity of a population (3) contributes to the repair of several types of DNA damage (A) Homologous Genetic Recombination Occurs in all prokaryotes and eukaryotes cells In prokaryotes, occurs during conjugation. (b) Separation of homologous pairs (Metaphase 1) Anaphase 1 In eukaryotes, occurs with the highest frequency during meiosis, a process in which diploid cells give rise to haploid cells destined to become gametes. First meiotic division Telophase 1 Prophase 2 DNA replication Diploid germ-line cell Second meiotic division Metaphase 2 (Anaphase 2) Telophase 2 Homologous recombination Prophase 1 Haploid gametes Recombination -> (1) Maintenance of genetic diversity (2) Facilitation of proper chromosome segregation during cell division in eukaryotes (-> promote evolution) 38 In eukaryotes Both on the same chromosome Nonsister chromatids (on homologous chromosomes) Chiasmata are the physical manifestation of homologous genetic recombination or crossing over Homologous junctions (Chiasmata or cross over points) (2) Facilitation of proper chromosome segregation during cell division in eukaryotes Holliday intermediate structure This type of structures is formed locally at the level of unwrapped DNA. A holliday intermediate formed between two bacterial plasmids in vivo. The physical positioning of the recombination structures is achieved by the formation of Holliday intermediate structures, crossover structures illustrating how different strands interact during the process of recombination. Branch migration 39 In eukaryotes Recombination during meiosis is initiated by double-strand breaks (“The double strand break repair model”) Double strand break converted into a double strand gap by exonucleases. 3’ ends are less degraded than 5’ ends leading to 3’ single strand extensions. 3’ 3’ An exposed 3’ end pairs with its complement in the intact homolog. The other strand of the duplex is displaced by branch migration. 3’ 3’ 3’ 3’ The invading 3’ end is extended by DNA polymerase and branch migration, eventually generating a DNA molecule with two crossovers called Holliday intermediates. 3’ Further DNA replication replaces the DNA missing from the site of the original doublestrand break. 4 1 2 3 8 5 6 7 (cuts at 2, 4, 6 and 8) Cleavage of the Holliday intermediates by specialized nucleases generates either of the two recombination products. Note that two other product sets are possible! (cuts at 1, 3, 6 and 8) 40 Note that homologous recombination mechanisms can vary in many details from one species to another! Most of the steps outlined in the previous model are generally present Another model for recombination RecBCD enzyme: a specific enzyme involved in homologous recombination (in E. coli) (5’)GCTGGTGG RecBCD enzyme -> nuclease and helicase activities. --> Binds free broken DNA ends --> Unwinds the DNA --> Degrades both strands of DNA until encountering a Chi sequence (~1000 seq/ genome). After that, it degrades only the 5’ end strand --> The 3’ end structure sequence is then used for subsequent recombination steps 41 RecA protein promotes the pairing of two DNA strands, the formation of Holliday intermediates and branch migration. RecA proteins form a supramolecular right handed helix with single or double stranded DNA RecA unit DNA axis Assembly of RecA is ATP dependent! RecA filament assembly is under the control of various proteins: Filament assembly is favored by various proteins (RecFOR). Rec X inhibits RecA filament extension while Dinl prevents disassembly. 42 The DNA strand-exchange reactions promoted by RecA protein in vitro -> A good model for illustrating the recombination activity of RecA filaments -> The exchange proceeds from 5’ to 3’ and occurs at a rate of 6bp/sec and can involved 3 or 4 strands 43 (3) DNA damage and its effect on DNA replication Can lead to error-prone translesion DNA synthesis (Study of recombination in prokaryotes) Recombinational DNA repair in case of stalled replication forks All aspects of DNA metabolism are involved to repair stalled replication forks. 44 RuvAB branch migration protein (RuvA+ RuvB) is a resolvase that binds to Holliday intermediates, displaces RecA and promotes branch migration at higher rates than does RecA. Resolvase RuvC specifically cleaves at holliday intermediates to generate fulllength, unbranched chromosome products. Origin-independent replication restart : the replication is reassembled with the aid of a complex of 7 proteins (PriA, B, and C and DnaB, C, G (primase) and T): the φX174 type primosome or replication restart primosome. Involvement of DNA Pol II and DNA Pol III. 45 Error-prone repair and SOS response: a desperation strategy Extensive DNA damage in bacteria triggers the induction of many distantly located genes No proofreading activity! No proofreading activity! DNA polymerase V -> can replicate past many DNA lesions= translesion replication (error-prone) UmuD protein cleaved -> UmuD’ (shorter product) UmuD’ + UmuC -> DNA pol V Error-prone repair and SOS response (Chapter 28 p.1130) The key regulatory elements are the RecA protein and LexA repressor (1) When DNA is extensively damaged, DNA replication is halted and the number of single-strand gaps in the DNA increases. (2) RecA protein binds to single stranded DNA, and activates its coprotease activity. (3) When interacting with LexA repressor, RecA induces LexA inactivation by promoting LexA own hydrolysis at a specific Ala-gly peptide bond. When LexA is inactivated, the SOS genes including RecA are induced. (RecA levels increase up to 100-fold) Note: SOS response can lead to the induction of replication of dormant lysogenic viruses. 46 (B) Site-specific recombination -- Recombination site sequences recognized by recombinases are partially asymmetric (nonpalindromic). -- The two recombination sites align in the same orientation (same order of nucleotides). -- If recombination sites on the same molecule -> inversion or deletion of the intervening DNA sequence. -- If recombination on different molecules recombination is intermolecular -> insertion of the DNA if one of the two molecules is circular. Recombination sites 47 (B) In site-specific recombination, the exchanges occur only at a particular DNA sequence Various functions (e.g. regulation of genes expression, promotion of programmed DNA rearrangement during embryonic development or in some viral and plasmid DNA replication) Site recombination system consists of a recombinase and unique DNA sequences, the recombination sites (20 to 200 bp). Recombinases can be viewed as site-specific endonucleases and ligases in one package. Example of a four-subunit integrase-class recombinase the Cre recombinase (encoded E.coli phage P1) bound to a Holliday structure recombination intermediate. Fundamental reaction pathway of recombinases (1) Recombinase subunits bind to the recombination site. One DNA strand in each DNA is cleaved at particular points within the sequence and becomes covalently linked to the recombinase through a phosphotyrosine bond (break a phosphodiester bond to make a phosphoester bond) No need of ATP! The cleaved strand is joined to new partners, forming a Holliday intermediate and (4) To complete the reaction, the process must be repeated at a second point within each of the recombination sites 1 2 1 2 4 1 34 2 1 3 (2) 2 (3) 4 34 3 1 2 4 3 Holliday intermediate 48 Site-specific recombination system encoded by the bacteriophage λ After infection of E. coli: Solution 1 The λ DNA replicates and produces more phage -> bacteria lysis Solution 2 The λ DNA integrates the bacterial chromosome -> dormant phage Site-specific recombination system encoded by the bacteriophage λ Phage recombinase = λ integrase (INT protein) -> attP shares only 15pb homologous to attB -> Integration requires INT and the Integration Host Factor (IHF) -> Excision requires INT, FIS (from bacteria) and XIS (Phage) 49 Complete chromosome replication can require site-specific recombination During recombinational DNA repair, the resolution of a Holliday junction by RuvC followed by completion of replication can give rise to either the usual two monomeric chromosomes or a contiguous dimeric chromosome. A specialized site-specific recombination system in E. coli, the XerCD system, converts the dimeric genome into two monomeric chromosomes, allowing chromosome segregation and cell division to proceed. 2x Mutations of chromosomes that can occur during recombination events 50 (C) DNA transposition Involves a short segment of DNA with the capacity to move from one location in a chromosome to another (first discovered in maize by Barbara McClintock, 1940s). Transposable elements are called transposons (“jumping genes”) Could be seen as the simplest molecular parasites. No DNA sequence homology usually required New location (target site) chosen randomly. -> could kill the cell -> tightly regulated, infrequent 5-10bp Two classes of transposons in bacteria (1) Insertion sequences (simple transposons) Contain sequences required for transposition genes for promoting the process (transposases)) (2) Complex transposons Contain one or more genes in addition to those required for transposition (e.g. genes of resistance for the host cell) The two general pathways for transposition in bacteria -> Direct or simple transposition -> Replicative transposition Cuts on both side of the transposon excise it Transposon linked to the target DNA Should be repaired Transposon moves to a new location 3’ hydroxyl attack of the transposon frees ends on the target DNA (catalyzed by transposase) The target phosphodiester bonds are staggered Replication fills in gap at each end (DNA polymerase and ligase) 51 Replicative transposition One cut on each end of the transposon Transposon linked to the target DNA The entire transposon is replicated -> Cointegrate intermediate Transposon moves to a new location 3’ hydroxyls of the transposon free ends attack the target DNA (catalyzed by transposase) Target phosphodiester bonds are staggered The cointegrate is converted to products by site-specific recombination (use of specialized recombinases) Example of programmed genetic rearrangement in eukaryotic cells : adaptive immune system Immunoglobulin genes are assembled by recombination Immunoglobulin genes are generated by recombination from separate gene segments. Recombination allows to produce an extraordinary diversity of antibodies from a limited set of genes (1000000 different antibodies with distinct binding sites). Variable regions Ig: 2 heavy chains 2 light chains (2 types kappa and lambda) Constant regions (constant within a class of Ig) (See Chapter 5) For heavy chain and kappa and lambda light chains, diversity is generated by a similar mechanism! 52 Example: recombination of the V and J segments of the human IgG kappa light chain V: 95 first aas of V chain J: 12 remaining aas of V chain C: encodes aas of constant region Recombination! ~ 300 V segments ~ 4 J segments 1200 possible V-J combinations As the recombination is not very precise, 2.5 x 1200= 3000 possible V-J combinations As ~ 5000 different heavy chains 3000 x 5000 = 1.5 x 107 possible IgG (High mutation rates within the variable regions during lymphocyte differentiation -> additional diversity) Each mature lymphocyte B produces only one Ig but numerous cells with different Ig. Mechanism of immunoglobulin gene rearrangement RSS: Recombination signal sequences RAG 1 and RAG 2: recombination activation gene proteins. Catalyze the formation of a double strand break between RSS and the V (or J) segments to be joined A second complex of proteins specialized for end joining repair of double strand breaks joins the V and J segments. Sequence structure of Intervening DNA is similar to the one found in most transposons! --> the system for producing antibody diversity may have evolved form an ancient cellular invasion of transposons! 53 ...
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