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2-13-2008 Recombination

2-13-2008 Recombination. 2-13-2008 Recombination. Overview of Recombination Requirements of Recombination Functions of Recombination Mechanism of Recombination and Holiday structure formation. Enzymes involved Chi-sites-function

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2-13-2008 Recombination

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  1. 2-13-2008 Recombination

  2. 2-13-2008 Recombination Overview of Recombination Requirements of Recombination Functions of Recombination Mechanism of Recombination and Holiday structure formation. Enzymes involved Chi-sites-function Examples of consequences of recombination and gene conversion: hin recombinase and Salmonella flagella,

  3. Four Requirements for Recombination • Identical or very similar DNA sequences in the cross over region. What would happen to the structure of the chromosome if this were not the case? • Complementary base pairing between double stranded DNA molecules. This ensures that recombination will occur only at these sequences. What would happen if this did not happen? • Recombination enzymes-the machinery of the recombination complex. • Heteroduplex formation-complementary base pairing between two DNA molecules in a synapse. This process occurs in all organisms capable of undergoing some kind of genetic exchange. Replication to fill in any gaps.

  4. Induction of Recombination • Phage infection • DNA damage, SOS response: uv irradiation, DNA damaging compounds, thymidine starvaton • Stalled replication forks-variety of reasons • Conjugal plasmid introduction-F and others • ?

  5. Figure 10.1

  6. Figure 10.3 Single strand invasion model

  7. Figure 10.4 Double strand break model

  8. Dr. Richard Losick, Harvard http://www.cfkeep.org/html/snapshot.php?id=41670106,

  9. Figure 10.5

  10. Definition of a Chi Site: Chi-sites were discovered by the lambda phage biologists. A mutant of lambda, called a red-gam mutant that lacks its own recombination systems, was used to infect wild type E.coli. Red-gam mutants are unable to replicate DNA normally. -small plaques were observed, but large plaques were also seen. -the only way for the phage to propagate is for recombination to occur with the chromosome at a high frequency -lambda DNA isolated from these plaques and sequenced -a sequence was identified 5’GCTGGTGG3’ that is not in wild type lambda -the sequence came from the E.coli chromosome What is the frequency of a 8-base pair sequence? 4-base: 44 256 5-base: 45 1024 6-base: 46 4096 8-base: 48 65536 Frequency of Chi-sites in the E.coli chromosome: 1 every 4-5kb

  11. Single stranded region formed by RecBDC now loaded with RecA protein for strand invasion. The D-loop is the displace strand

  12. Figure 10.7

  13. Figure 10.6

  14. Figure 10.8

  15. RuvABC complex -a large doughnut

  16. Holiday Structure 1964 Robin Holiday www.mun.ca/biochem/courses/3107/Lectures/Topics/Recombination_holliday.html

  17. Two diagrams of of Salmonella flagellar phase variation -fliB (H2) locked on mutants-non virulent -fliC (H1) locked on mutants-virulent

  18. Importance of RecA for pathogens-Salmonella Wild type Lethal Dose for mice Wt: 10 cfu recA: 50,000+ cfu Cfu recA Time-hours recA mutants cannot survive in host macrophages What does this tell about the environment of the host?

  19. Streptococcus M-protein -Streptococcus-GroupA Strep synthesize surface protein called M-protein -There are 80 variations of M-protein made -Production of variants results from slip-strand replication/recombination within the M-protein coding cassettes. -Production of multiple M-types assist in evading host defenses.

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