Welcome Each of You to My Molecular Biology Class

1. Welcome Each of You to My Molecular Biology Class

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. 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. 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. Molecular Biology Course Chapter 11 Site-Specific Recombination & Transposition of DNA

6. There are genetic processes that rearrange DNA sequences and thus lead to a more dynamic genome structure

7. Two classes of genetic recombination • Conservative site-specific recombination (CSSR): recombination between two defined sequence elements • Transpositional recombination (Transposition): recombination between specific sequences and nonspecific DNA sites

8. Figure 11-1

9. OUTLINE • Conservative Site-Specific Recombination • Biological Roles of Site-Specific Recombination • Transposition • Examples of Transposable Elements and Their Regulation • V(D)J Recombination

10. Topic 1: Conservative Site-Specific Recombination • Exchange of non-homologous sequences at specific DNA sites(what) • Mediated by proteins that recognize specific DNA sequences. (how)

11. Conservative Site-Specific Recombination 1-1 Site-specific recombination occurs at specific DNA sequences in the target DNA • CSSR (conserved site-specific recombination) is responsible for many reactions in which a defined segment of DNA is rearranged.

12. CSSR can generate three different types of DNA rearrangements Figure 11-3

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. 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. Figure 11-4 Structures involved in CSSR

16. Conservative Site-Specific Recombination 1-2 Site-specific recombinases cleave and rejoin DNA using a covalent protein-DNA intermediate • Serine Recombinases • Tyrosine Recombinases Figure 11-5

17. Conservative Site-Specific Recombination 1-3 Serine recombinases introduce double-stranded breaks in DNA and then swap strands to promote recombination

18. Conservative Site-Specific Recombination Figure 11-6

19. Conservative Site-Specific Recombination 1-4 Tyrosine recombinases break and rejoin one pair of DNA strands at a time

20. Figure 11-7

21. Conservative Site-Specific Recombination 1-5Structure of tyrosine recombinases bound to DNA reveal the mechanism of DNA exchange • Cre is a tyrosine recombinase • Cre is an phage P1-encoded protein, functioning to circularize the linear phage genome during infection • The recombination sites of Cre is lox sites. Cre-lox is sufficient for recombination

22. Figure 11-8

24. Topic 2 Biological roles of site-specific recombination

25. Many phage insert their DNA into the host chromosome during infection using this recombination mechanism. Example: l phage • Alter gene expression. Example: Salmonella Hin recombinase • Maintain the structural integrity of circular DNA molecules during cycles of DNA replication. Example: resolvase that resolves dimer to monomer

26. The general themes of site-specific recombination All reactions depend critically on the assembly of the recombinase protein on the DNA and bring together of the two recombination sites 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.

27. Biological roles of site-specific recombination 2-1 l integrase promotes the integration and Excision of a Viral Genome into the Host Cell Chromosome The outcome of l bacteriophage infection of a host bacterium • Establishment of the lysogenic state: requires the integration of phage DNA into host chromosome • 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.

28. Figure 11-2: l 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.

29. Phage genome Crossover regions Bacterial genome Int (l-encoded integrase) Xis (l-encoded excisionase) IHF (integration host factor encoded by bacteria) Figure 11-9

30. l-encoded integrase (Int) • catalyzes recombination between two attachment (att) sites. attP site is on the phage DNA and attB site is on the bacterial genome • Is a tyrosine recombinase, and the mechanism of strand exchange is similar to that catalyzed by Cre recombinase. • 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).

31. Biological roles of site-specific recombination 2-3 The Hin recombinase inverts a segment of DNA allowing expression of alternative genes The Salmonella Hin recombinase inverts a segment of the bacterial chromosome to allow expression of two alternative sets of genes, which is known as an example of programmed rearrangements common in bacteria. Hin inversion is used to help the bacteria evade the host immune system. Hin is a serine recombinase

32. The genes controlled by Hin inversion encode two alternative forms of flagellin, the protein component of the flegellar fillament. Flagella are on the surface of the bacteria and thus a common target for the immune system. By using Hin inversion to switch between these two alternative forms, some bacterial individuals can aviod recognition by host immune system Figure 11-11 Salmonella (showing flegella) invading cultured human cells.

33. fljB encodes H2 flagellin fljA encodes a transcriptional repressor of H1 flagellin Figure 11-11 Hin inversion

34. Biological roles of site-specific recombination 2-5 Recombinase converts multimeric circular DNA molecules into monomers The chromosomes of most bacteria, plasmids and some viral genomes are circular. 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. Site-specific recombinases also called resolvases catalyze such a process.

35. Figure 11-14 Circular DNA molecules can form multimers

36. Xer recombinase is a tyrosine recombinase and catalyzes the monomerization of bacterial chromosomes and of many bacterial plasmids. Xer is a heterotetramer containing two subunits of XerC and two subunits of XerD. XerC and XerD recognize different DNA sequence.

37. Figure 11-15 The dimer only resolves when XerD is activated by the presence of FtsK

38. Topic 3 Transposition (转座) • Transposition is a specific form of genetic recombination that moves certain genetic elements from one DNA site to another. • These mobile genetic elements are called transposable elements or transposons. • 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.

39. 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.

40. 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. Box 11-3 Example of corn cob showing color variegation due to transposition

41. 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 plant genomes are very rich in functional transposons

42. 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.

43. 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.

44. 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 chromosomesand their behaviors at mitosis and meiosis. Chromosomes then became a source of fascination as they were known to be the bearers of "heritable factors". 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.

45. 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.

46. 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.

47. 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.

48. Born 1902, Brooklyn, New York • B.A. 1923, Cornell University • Ph.D. 1927, Cornell University, Botany • 1927-1931, Instructor in Botany, Cornell University • 1931-1933, Fellow, National Research Council • 1933-1934, Fellow, Guggenheim Foundation • 1934-1936, Research Associate, Cornell University • 1936-1941, Assistant Professor, University of Missouri • 1942-1967, Staff member, Carnegie Institution of Washington's Department of Genetics, Cold Spring Harbor, NY • 1967-1992, Distinguished Service Member, CIW Department of Genetics, Cold Spring Harbor • 1944,Member, National Academy of Sciences • 1945, President, Genetics Society of America • 1967, Kimber Medal • 1970, National Medal of Science • 1981, Lasker Award • 1983,Nobel Prize in Physiology or Medicine

49. The biological relevance of transposons • 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) • The genetic recombination mechanisms of transposition are also used for other functions, such as integration of some virus into the host genome and some DNA rearrangement to alter gene expression [V(D)J recombination].

50. 3-(1-6) There are three principle classes of transposable elements Transposition • DNA transposons • Viral-like retrotransposons including the retrovirus, which are also called LTR retrotransposons • Poly-A retrotransposons, also called nonviral retrotransposons.