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Welcome Each of You to My Molecular Biology Class
Molecular Biology of the Gene, 5/E   --- Watson et al. (2004) Part I: Chemistry and Genetics  Part II: Maintenance of the ...
Part II: Maintenance of the Genome Dedicated to the  structure  of DNA and the  processes  that  propagate ,  maintain  an...
Ch 6: The  structures  of DNA and RNA  Ch 7: Chromosomes, chromatins and the nucleosome Ch 8: The  replication  of DNA Ch ...
Chapter 11  Site-Specific Recombination & Transposition of DNA <ul><li>Molecular Biology Course </li></ul>
Although DNA replication, repair, homologous recombination occur with high fidelity to ensure the  genome identity  betwee...
<ul><li>Two classes of genetic recombination for DNA rearrangement: </li></ul><ul><li>Conservative site-specific recombina...
Figure 11-1
OUTLINE <ul><li>Conservative Site-Specific Recombination </li></ul><ul><li>Biological Roles of Site-Specific Recombination...
Topic 1: Conservative Site-Specific Recombination <ul><li>Exchange of non-homologous sequences at specific DNA sites (what...
<ul><li>CSSR  (conserved site-specific recombination) is responsible for many reactions in which  a defined segment of DNA...
CSSR can generate  three  different types of DNA rearrangements <ul><li>Figure 11-3 </li></ul>
If the two sites at which recombination will take place are  oriented oppositely to one another in the same DNA molecule  ...
If the two sites at which recombination will take place  are oriented in the same direction in the same DNA molecule,  the...
<ul><li>Figure 11-4 Structures involved in CSSR </li></ul>
<ul><li>Serine Recombinases </li></ul><ul><li>Tyrosine Recombinases </li></ul>Conservative Site-Specific Recombination 1-2...
The covalent protein-DNA intermediate conserves the energy of the cleaved phosphodiester bond within the protein-DNA linka...
Conservative Site-Specific Recombination 1-3  Serine recombinases introduce double-stranded breaks in DNA and then swap st...
Conservative Site-Specific Recombination Figure 11-6
Conservative Site-Specific Recombination 1-4  Tyrosine recombinases break and rejoin one pair of DNA strands at a time
Figure 11-7
Conservative Site-Specific Recombination 1-5   Structure  of tyrosine recombinases bound to DNA reveal the  mechanism  of ...
Figure 11-8
 
Topic 2 Biological roles of site-specific recombination
<ul><li>Many  phage insert their DNA  into the host chromosome during infection using this recombination mechanism.  Examp...
<ul><li>All reactions depend critically on the  assembly  of the recombinase protein on the DNA and bring together of the ...
2-1& 2    integrase works with IHF and Xis to integrate/excise the phage genome into/from the bacterial chromosome <ul><l...
<ul><li>Figure 11-2:   genome integration.  Recombination always occurs at exactly the same sequence within two recombin...
Bacterial genome Phage genome Crossover regions Int  -encoded integrase) Xis (  -encoded excisionase)  IHF  integrat...
<ul><li> -encoded integrase (Int) </li></ul><ul><li>catalyzes recombination between two attachment ( att ) sites.  attP  ...
2-5 Recombinase  converts multimeric circular DNA molecules into monomers <ul><li>The chromosomes of most bacteria, plasmi...
Figure 11-14 Circular DNA molecules can form multimers
Xer recombinase catalyzes the monomerization of bacterial chromosomes and of many bacterial plasmids. Xer recombinase is a...
Figure 11-15  The dimer only resolves when XerD is activated by the presence of FtsK
Topic 3 Transposition ( 转座 ) <ul><li>Transposition  is a specific form of genetic recombination that moves certain genetic...
<ul><li>FIGURE 11-17  Transposition of a mobile genetic element to a new site in host DNA,   which occurs with or without ...
<ul><li>Because transposition has little sequence selectivity in their choice of insertion sites, the transposons can inse...
<ul><li>It was actually the ability of transposable elements to break chromosomes that first came to McClintock attention ...
Discovery of Transposition Barbara McClintock In the  fall of 1921  I attended  the only course in genetics open to underg...
The results of these studies provided a solid conceptual framework into which subsequent results could be fitted. Neverthe...
At the time I was taking the undergraduate genetics course, I was enrolled in a  cytology course  given by  Lester W. Shar...
After completing requirements for the Ph.D. degree in the spring of 1927,  I remained at Cornell  to initiate studies aime...
Rhoades  had taken a Masters degree at the California Institute of Technology and  was well versed in the newest findings ...
Over the years,  members of this group have retained the warm personal relationship that our early association generated. ...
<ul><li>Born 1902, Brooklyn, New York  </li></ul><ul><li>B.A. 1923, Cornell University  </li></ul><ul><li>Ph.D. 1927, Corn...
You, CLS students of Wuhan University, get to learn to enjoy science/to be interested in science, but not to enjoy good sc...
The biological relevance of transposons <ul><li>Transposons are present in the genomes of all life-forms.  (1) transposon-...
2. The genetic recombination mechanisms of transposition are also  used for other functions  than the movement of transpos...
3-(1-6)  There are three principle classes of transposable elements <ul><li>DNA transposons </li></ul><ul><li>Viral-like r...
FIGURE 11-19  Genetic organization of the three classes of transposable elements
3-2  DNA transposons carry a  transposase  gene, flanked by  recombination sites <ul><li>Recombination sites  are at the t...
3-3  Transposons exist as both autonomous and nonautonomous elements <ul><li>Autonomous transposons : carry a pair of term...
3-4  Viral-like retrotransposons  and retroviruses carry terminal repeat sequences and two genes important for recombinati...
3-5  Poly-A retrotransposons  look like genes <ul><li>Do not have the terminal inverted repeats.  </li></ul><ul><li>On end...
3-(7-9)  DNA transposition by a cut-and-paste mechanism (non-replicative mechanism) <ul><li>Multimers of transposase binds...
<ul><li>The  transposase  first cleaves one DNA strand at each end of the transposon, resulting in free 3’-OH groups </li>...
<ul><li>The  3’OH ends  of the transposon attack the DNA phosphodiester bonds at the sites of the new insertion/target DNA...
FIGURE 11-20 The cut-and-paste mechanism of transposition One-step transesterification
3-10  DNA transposition by a replicative mechanism/replicative transposition <ul><li>The mechanism is similar to the cut-a...
<ul><li>The 3’OH ends of the transposon DNA are then joined to the target sites by the  DNA strand transfer  reaction, res...
FIGURE 11-22 Replicative transposition
 
3-11  Viral-like Retrotransposons & Retroviruses  move using an RNA intermediate <ul><li>The mechanism is similar to the D...
<ul><li>The RNA is then  reverse transcribed  to cDNA (dsDNA) that is free from any flanking host DNA sequences, resulting...
Figure 11-23 Mechanism of retroviral integration and transposition of viral-like retrotransposons.
3-12  DNA transposases and retroviral integrases are members of a  protein superfamily Transposition FIGURE 11-24 Similari...
FIGURE 11-24 Similarity of catalytic domains of transposases and integrases. (b) Scematic of the domain organization of th...
3-13  Poly-A Retrotransposition  move by a “reverse splicing” mechanism <ul><li>Using an RNA intermediate but a different ...
<ul><li>The protein-RNA complex reenters the nuclease and associate with the chromosomal DNA </li></ul><ul><li>The endonuc...
 
 
Key points <ul><li>Conservative Site-Specific Recombination  (concept, three types, mechanisms-serine and  tyrosine  recom...
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  1. 1. Welcome Each of You to My Molecular Biology Class
  2. 2. Molecular Biology of the Gene, 5/E --- Watson et al. (2004) Part I: Chemistry and Genetics Part II: Maintenance of the Genome Part III: Expression of the Genome Part IV: Regulation Part V: Methods
  3. 3. Part II: Maintenance of the Genome Dedicated to the structure of DNA and the processes that propagate , maintain and alter it from one cell generation to the next
  4. 4. Ch 6: The structures of DNA and RNA Ch 7: Chromosomes, chromatins and the nucleosome Ch 8: The replication of DNA Ch 9: The mutability and repair of DNA Ch 10: Homologous recombination at the molecular level Ch 11: Site-specific recombination and transposition of DNA
  5. 5. Chapter 11 Site-Specific Recombination & Transposition of DNA <ul><li>Molecular Biology Course </li></ul>
  6. 6. Although DNA replication, repair, homologous recombination occur with high fidelity to ensure the genome identity between generations, there are genetic processes that rearrange DNA sequences and thus lead to a more dynamic genome structure
  7. 7. <ul><li>Two classes of genetic recombination for DNA rearrangement: </li></ul><ul><li>Conservative site-specific recombination (CSSR): recombination between two defined sequence elements </li></ul><ul><li>Transpositional recombination (Transposition): recombination between specific sequences and nonspecific DNA sites </li></ul>
  8. 8. Figure 11-1
  9. 9. OUTLINE <ul><li>Conservative Site-Specific Recombination </li></ul><ul><li>Biological Roles of Site-Specific Recombination (  phage integration/excision, multimeric genome resolution) </li></ul><ul><li>Transposition ( concepts, learning from B. McClintock, DNA tranposons. Viral-like retrotransposons/retroviruses, poly-A retrotransposons) </li></ul>
  10. 10. Topic 1: Conservative Site-Specific Recombination <ul><li>Exchange of non-homologous sequences at specific DNA sites (what) </li></ul><ul><li>Mediated by proteins that recognize specific DNA sequences. (how) </li></ul>
  11. 11. <ul><li>CSSR (conserved site-specific recombination) is responsible for many reactions in which a defined segment of DNA is rearranged . </li></ul>Conservative Site-Specific Recombination 1-1 Site-specific recombination occurs at specific DNA sequences in the target DNA
  12. 12. CSSR can generate three different types of DNA rearrangements <ul><li>Figure 11-3 </li></ul>
  13. 13. If the two sites at which recombination will take place are oriented oppositely to one another in the same DNA molecule then the site-specific reacombination results in inversion of the segment of DNA between the two recombination sites recombination at inverted repeats causes an inversion
  14. 14. If the two sites at which recombination will take place are oriented in the same direction in the same DNA molecule, then the segment of DNA between the two recombinogenic sites is deleted from the rest of the DNA molecule and appears as a circular molecule. Insertion is the reverse reaction of the deletion recombination at direct repeats causes a deletion
  15. 15. <ul><li>Figure 11-4 Structures involved in CSSR </li></ul>
  16. 16. <ul><li>Serine Recombinases </li></ul><ul><li>Tyrosine Recombinases </li></ul>Conservative Site-Specific Recombination 1-2 Site-specific recombinases cleave and rejoin ( join ) DNA using a covalent protein-DNA intermediate Figure 11-5
  17. 17. The covalent protein-DNA intermediate conserves the energy of the cleaved phosphodiester bond within the protein-DNA linkage, which allows the cleaved DNA strands to be rejoined/resealed by reversal of the the cleavage process This mechanistic feature contributes the “ conservative ” to the CSSR name, because every DNA bond that is broken during the reaction is resealed by the recombinase without consuming any external energy.
  18. 18. Conservative Site-Specific Recombination 1-3 Serine recombinases introduce double-stranded breaks in DNA and then swap strands to promote recombination
  19. 19. Conservative Site-Specific Recombination Figure 11-6
  20. 20. Conservative Site-Specific Recombination 1-4 Tyrosine recombinases break and rejoin one pair of DNA strands at a time
  21. 21. Figure 11-7
  22. 22. Conservative Site-Specific Recombination 1-5 Structure of tyrosine recombinases bound to DNA reveal the mechanism of DNA exchange <ul><li>Cre is a tyrosine recombinase </li></ul><ul><li>Cre is an phage P1-encoded protein, functioning to circularize the linear phage genome during infection </li></ul><ul><li>The recombination sites of Cre is lox sites. Cre- lox is sufficient for recombination </li></ul><ul><li>Read Box11-1 for Cre application </li></ul>
  23. 23. Figure 11-8
  24. 25. Topic 2 Biological roles of site-specific recombination
  25. 26. <ul><li>Many phage insert their DNA into the host chromosome during infection using this recombination mechanism. Example:  phage </li></ul><ul><li>Alter gene expression . Example: Salmonella Hin recombinase (the details are not discussed in the class) </li></ul><ul><li>Maintain the structural integrity of circular DNA molecules during cycles of DNA replication. Example: resolvase that resolves dimer to monomer </li></ul>
  26. 27. <ul><li>All reactions depend critically on the assembly of the recombinase protein on the DNA and bring together of the two recombination sites </li></ul><ul><li>Some recombination requires only the recombinase and its recognition sequence for such an assembly; some requires accessory proteins including Architectural Proteins that bind specific DNA sequences and bend the DNA. </li></ul>The general themes of site-specific recombination
  27. 28. 2-1& 2  integrase works with IHF and Xis to integrate/excise the phage genome into/from the bacterial chromosome <ul><li>The outcome of  bacteriophage infection of a host bacterium </li></ul><ul><li>Establishment of the lysogenic state : requires the integration of phage DNA into host chromosome </li></ul><ul><li>lytic growth is the growth stage of multiplication of the independent phage DNA that requires the excision of the integrated phage DNA from the host genome. </li></ul>Biological roles of site-specific recombination
  28. 29. <ul><li>Figure 11-2:  genome integration. Recombination always occurs at exactly the same sequence within two recombination sites, one on the phage DNA, and the other on the bacterial DNA. </li></ul>
  29. 30. Bacterial genome Phage genome Crossover regions Int  -encoded integrase) Xis (  -encoded excisionase) IHF  integration host factor encoded by bacteria) Figure 11-9
  30. 31. <ul><li> -encoded integrase (Int) </li></ul><ul><li>catalyzes recombination between two attachment ( att ) sites. attP site is on the phage DNA and attB site is on the bacterial genome </li></ul><ul><li>Is a tyrosine recombinase , and the mechanism of strand exchange is similar to that catalyzed by Cre recombinase. </li></ul><ul><li>Requires accessory proteins to assemble the integrase on the att sites. Both IHF and Xis are architectural proteins. IHF binds to DNA to bring together the Int recognition sites. Xis binds to the integrated att sites to stimulate excision and to inhibit integration (see 2-2 ). </li></ul>
  31. 32. 2-5 Recombinase converts multimeric circular DNA molecules into monomers <ul><li>The chromosomes of most bacteria, plasmids and some viral genomes are circular. </li></ul><ul><li>During the process of homologous recombination, these circular DNA sometimes form dimers and even multimeric forms, which can be can be converted back into monomer by site specific recombination. </li></ul><ul><li>Site-specific recombinases also called resolvases catalyze such a process. </li></ul>Biological roles of site-specific recombination
  32. 33. Figure 11-14 Circular DNA molecules can form multimers
  33. 34. Xer recombinase catalyzes the monomerization of bacterial chromosomes and of many bacterial plasmids. Xer recombinase is a member of the tyrosine recombinase family Xer is a heterotetramer containing two subunits of XerC and two subunits of XerD. Both XerC and XerD are tyrosine recombinases but recognize different DNA sequence. The recombination sites in bacterial chromosomes, called dif sites have recognition sites for both XerC and XerD.
  34. 35. Figure 11-15 The dimer only resolves when XerD is activated by the presence of FtsK
  35. 36. Topic 3 Transposition ( 转座 ) <ul><li>Transposition is a specific form of genetic recombination that moves certain genetic elements from one DNA site to another. </li></ul><ul><li>These mobile genetic elements are called transposable elements or transposons. </li></ul><ul><li>Movement occurs through recombination between the DNA sequences at the ends of the transposons and a sequence in the host DNA with little sequence selectivity. </li></ul>
  36. 37. <ul><li>FIGURE 11-17 Transposition of a mobile genetic element to a new site in host DNA, which occurs with or without duplication of the element. </li></ul>
  37. 38. <ul><li>Because transposition has little sequence selectivity in their choice of insertion sites, the transposons can insert within genes or regulatory sequence of a gene , which results in the completely disruption of gene function or the alteration of the expression of a gene. These disruption leads to the discovery of transposable elements by Barbara McClintock. </li></ul>Box 11-3 Example of corn cob showing color variegation due to transposition
  38. 39. <ul><li>It was actually the ability of transposable elements to break chromosomes that first came to McClintock attention (late 1940s). She found that some maize ( 玉米 ) strains experienced frequent chromosome-broken, and the “hotspots” for chromosome breaks varied among different strains and among different chromosomal locations in the descendents ( 后代 ) of an individual plant, which leads to the concept that genetic elements could move/transpose </li></ul>plant genomes are very rich in functional transposons
  39. 40. Discovery of Transposition Barbara McClintock In the fall of 1921 I attended the only course in genetics open to undergraduate students at Cornell University. It was conducted by C. B. Hutchison, then a professor in the Department of Plant Breeding, College of Agriculture, who soon left Cornell to become Chancellor of the University of California at Davis, California. Relatively few students took this course and most of them were interested in pursuing agriculture as a profession. Genetics as a discipline had not yet received general acceptance . Only twenty-one years had passed since the rediscovery of Mendel's principles of heredity. Genetic experiments, guided by these principles, expanded rapidly in the years between 1900 and 1921.
  40. 41. The results of these studies provided a solid conceptual framework into which subsequent results could be fitted. Nevertheless, there was reluctance on the part of some professional biologists to accept the revolutionary concepts that were surfacing. This reluctance was soon dispelled as the logic underlying genetic investigations became increasingly evident. When the undergraduate genetics course was completed in January 1922, I received a telephone call from Dr. Hutchison. He must have sensed my intense interest in the content of his course because the purpose of his call was to invite me to participate in the only other genetics course given at Cornell. It was scheduled for graduate students . His invitation was accepted with pleasure and great anticipations. Obviously, this telephone call cast the die for my future . I remained with genetics thereafter .
  41. 42. At the time I was taking the undergraduate genetics course, I was enrolled in a cytology course given by Lester W. Sharp of the Department of Botany. His interests focused on the structure of chromosomes and their behaviors at mitosis and meiosis. Chromosomes then became a source of fascination as they were known to be the bearers of &quot;heritable factors&quot;. By the time of graduation, I had no doubts about the direction I wished to follow for an advanced degree. It would involve chromosomes and their genetic content and expressions, in short, cytogenetics. This field had just begun to reveal its potentials. I have pursued it ever since and with as much pleasure over the years as I had experienced in my undergraduate days.
  42. 43. After completing requirements for the Ph.D. degree in the spring of 1927, I remained at Cornell to initiate studies aimed at associating each of the ten chromosomes comprising the maize complement with the genes each carries. With the participation of others , particularly that of Dr. Charles R. Burnham, this task was finally accomplished. In the meantime, however, a sequence of events occurred of great significance to me . It began with the appearance in the fall of 1927 of George W. Beadle (a Nobel Laureate) at the Department of Plant Breeding to start studies for his Ph.D. degree with Professor Rollins A. Emerson . Emerson was an eminent geneticist whose conduct of the affairs of graduate students was notably successful, thus attracting many of the brightest minds. In the following fall, Marcus M. Rhoades arrived at the Department of Plant Breeding to continue his graduate studies for a Ph.D. degree, also with Professor Emerson.
  43. 44. Rhoades had taken a Masters degree at the California Institute of Technology and was well versed in the newest findings of members of the Morgan group working with Drosophila. Both Beadle and Rhoades recognized the need and the significance of exploring the relation between chromosomes and genes as well as other aspects of cytogenetics. The initial association of the three of us, followed subsequently by inclusion of any interested graduate student, formed a close-knit group eager to discuss all phases of genetics, including those being revealed or suggested by our own efforts. The group was self-sustaining in all ways. For each of us this was an extraordinary period. Credit for its success rests with Professor Emerson who quietly ignored some of our seemingly strange behaviors .
  44. 45. Over the years, members of this group have retained the warm personal relationship that our early association generated. The communal experience profoundly affected each one of us. The events recounted above were, by far, the most influential in directing my scientific life.
  45. 46. <ul><li>Born 1902, Brooklyn, New York </li></ul><ul><li>B.A. 1923, Cornell University </li></ul><ul><li>Ph.D. 1927, Cornell University, Botany </li></ul><ul><li>1927-1931, Instructor in Botany, Cornell University </li></ul><ul><li>1931-1933, Fellow, National Research Council </li></ul><ul><li>1933-1934, Fellow, Guggenheim Foundation </li></ul><ul><li>1934-1936, Research Associate, Cornell University </li></ul><ul><li>1936-1941 , Assistant Professor, University of Missouri </li></ul><ul><li>1942-1967, Staff member, Carnegie Institution of Washington's Department of Genetics, Cold Spring Harbor, NY </li></ul><ul><li>1967-1992, Distinguished Service Member, CIW Department of Genetics, Cold Spring Harbor </li></ul><ul><li>1944, Member, National Academy of Sciences </li></ul><ul><li>1945, President, Genetics Society of America </li></ul><ul><li>1967, Kimber Medal </li></ul><ul><li>1970, National Medal of Science </li></ul><ul><li>1981, Lasker Award </li></ul><ul><li>1983, Nobel Prize in Physiology or Medicine </li></ul>
  46. 47. You, CLS students of Wuhan University, get to learn to enjoy science/to be interested in science, but not to enjoy good scores.
  47. 48. The biological relevance of transposons <ul><li>Transposons are present in the genomes of all life-forms. (1) transposon-related sequences can make up huge fractions of the genome of an organism (50% of human and maize genome). (2) the transposon content in different genomes is highly variable (Fig 11-18). </li></ul>
  48. 49. 2. The genetic recombination mechanisms of transposition are also used for other functions than the movement of transposons, such as integration of some virus into the host genome and some DNA rearrangement to alter gene expression [V(D)J recombination].
  49. 50. 3-(1-6) There are three principle classes of transposable elements <ul><li>DNA transposons </li></ul><ul><li>Viral-like retrotransposons including the retrovirus, which are also called LTR retrotransposons </li></ul><ul><li>Poly-A retrotransposons , also called nonviral retrotransposons. </li></ul>Transposition
  50. 51. FIGURE 11-19 Genetic organization of the three classes of transposable elements
  51. 52. 3-2 DNA transposons carry a transposase gene, flanked by recombination sites <ul><li>Recombination sites are at the two ends of the transposon and are inverted repeated sequences varying in length from 25 to a few hundred bp. </li></ul><ul><li>The recombinase responsible for transposition are usually called transposases or integrases. </li></ul><ul><li>Sometimes they carry a few additional genes . Example, many bacterial DNA transposons carry antibiotic resistance gene. </li></ul>Transposition
  52. 53. 3-3 Transposons exist as both autonomous and nonautonomous elements <ul><li>Autonomous transposons : carry a pair of terminal inverted repeats and a transposase gene ; function independently </li></ul><ul><li>Nonautonomous transposons : carry the terminal inverted repeats but not the functional transposase ; need the transposase encode d by autonomous transposons to enable transposition </li></ul>Transposition
  53. 54. 3-4 Viral-like retrotransposons and retroviruses carry terminal repeat sequences and two genes important for recombination <ul><li>I nverted t erminal repeat sequences for recombinase binding are embedded within long terminal repeats (LTRs) , being organized on the two ends of the elements as direct repeats . </li></ul><ul><li>reverse transcriptase (RT ) , us ing an RNA template to synthesize DNA . </li></ul><ul><li>integrase (the transposase ) </li></ul>Transposition
  54. 55. 3-5 Poly-A retrotransposons look like genes <ul><li>Do not have the terminal inverted repeats. </li></ul><ul><li>On end is called 5’ UTR ( untranslated region ), the other end is 3’ UTR followed by a stretch of A-T base pairs called the poly-A sequence. Flanked by short target site duplication. </li></ul><ul><li>Carry two genes. ORF1 encodes an RNA-binding proteins. ORF2 encodes a protein with both reverse transcriptase (RT) and endonuclease activity. Truncated elements lacking complete 5’ UTR?? </li></ul>Transposition
  55. 56. 3-(7-9) DNA transposition by a cut-and-paste mechanism (non-replicative mechanism) <ul><li>Multimers of transposase binds to the terminal inverted repeats of the transposons, and bring two ends together to form a stable protein-DNA complex called the synaptic complex/ transpososome . </li></ul><ul><li>This complex ensures the DNA cleavage and joining reaction, which is called strand transfer and is similar to the recombinase </li></ul>Transposition
  56. 57. <ul><li>The transposase first cleaves one DNA strand at each end of the transposon, resulting in free 3’-OH groups </li></ul><ul><li>Different transposons use different mechanism to cleave the “second” strands , resulting in 5’ ends at the transposons. The mechanism including using a secondary enzyme (Tn 7 ), using an unusual DNA transesterification mechanism to generate a DNA hairpin structure subsequently resolved by transposases (Tn 10 , Tn 5 ) (3-9, Fig 11-21 ) </li></ul>
  57. 58. <ul><li>The 3’OH ends of the transposon attack the DNA phosphodiester bonds at the sites of the new insertion/target DNA, resulting in transposon insertion. This DNA rejoining reactions occurs by a one-step transesterification reaction called DNA strand transfer . </li></ul><ul><li>The intermediate with two nicks is finished by gap repair . The “old” insertion site having a double-stranded break are repaired by DSB repair ( 3-8 ) </li></ul>
  58. 59. FIGURE 11-20 The cut-and-paste mechanism of transposition One-step transesterification
  59. 60. 3-10 DNA transposition by a replicative mechanism/replicative transposition <ul><li>The mechanism is similar to the cut-and-paste transposition. </li></ul><ul><li>The assembly of the transposase protein on the two ends of the transposon DNA to generate the transpososome . </li></ul><ul><li>The transposase first cleaves one DNA strand at each end of the transposon, resulting in two 3’OH ends. BUT NO cleavage occurs at the second strand. </li></ul>Transposition
  60. 61. <ul><li>The 3’OH ends of the transposon DNA are then joined to the target sites by the DNA strand transfer reaction, resulting in a doubly branched DNA molecule . </li></ul><ul><li>The two branches within this intermediate have the structure of a replication fork, which recruits the replication proteins for strand synthesis. As a result, the donor DNA is duplicated in the host DNA. </li></ul><ul><li>Replicative transposition frequently causes chromosomal inversions and deletions that can be highly detrimental ( 有害的 ) to the host cell. </li></ul>
  61. 62. FIGURE 11-22 Replicative transposition
  62. 64. 3-11 Viral-like Retrotransposons & Retroviruses move using an RNA intermediate <ul><li>The mechanism is similar to the DNA transposons (Cut-and-Paste) . The major difference is the involvement of an RNA intermediate. </li></ul><ul><li>Transcription of the retrotransposon (or retroviral) DNA sequence into RNA by cellular RNA polymerase, which is initiated at a promoter sequence within one of the LTRs. </li></ul>Transposition
  63. 65. <ul><li>The RNA is then reverse transcribed to cDNA (dsDNA) that is free from any flanking host DNA sequences, resulting in the excised form of transposon </li></ul><ul><li>Integrase assembles on the ends of cDNA and cleaves a few nucleotides off the 3’ends, generating 3’OHs. </li></ul><ul><li>Integrase inserts the transposon into target site using the DNA strand transfer reaction. </li></ul><ul><li>Gap repair and ligation complete the recombination and generate target-site duplications. </li></ul>
  64. 66. Figure 11-23 Mechanism of retroviral integration and transposition of viral-like retrotransposons.
  65. 67. 3-12 DNA transposases and retroviral integrases are members of a protein superfamily Transposition FIGURE 11-24 Similarity of catalytic domains of transposases and integrases. (a) structure of the conserved core domains of three transposases and intergrase MuA Tn5 RSV integrase
  66. 68. FIGURE 11-24 Similarity of catalytic domains of transposases and integrases. (b) Scematic of the domain organization of the above three proteins
  67. 69. 3-13 Poly-A Retrotransposition move by a “reverse splicing” mechanism <ul><li>Using an RNA intermediate but a different mechanism from that of the viral-like retrotransposons </li></ul><ul><li>The mechanism used is called target site primed reverse transcription . </li></ul><ul><li>Transcription of the integrated DNA </li></ul><ul><li>The newly synthesized RNA is exported to cytoplasm to produce ORF1 and ORF2 proteins, which remain to bind the RNA </li></ul>Transposition
  68. 70. <ul><li>The protein-RNA complex reenters the nuclease and associate with the chromosomal DNA </li></ul><ul><li>The endonuclease activity of ORF2 introduce a nick on the chromosomal DNA at the T-rich sites. </li></ul><ul><li>The 3’OH generated on the target DNA serves as the primer for reverse transcription of the element RNA (ORF2) </li></ul>
  69. 73. Key points <ul><li>Conservative Site-Specific Recombination (concept, three types, mechanisms-serine and tyrosine recombinases) </li></ul><ul><li>Biological Roles of Site-Specific Recombination (  phage integration/excision, multimeric genome resolution) </li></ul><ul><li>Transposition ( concepts, learning from B. McClintock, DNA tranposons. Viral-like retrotransposons/retroviruses, poly-A retrotransposons) </li></ul>
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