2-13-2008 Recombination

2-13-2008 Recombination

1. Overview of Recombination

2. Requirements of Recombination

3. Functions of Recombination

4. Mechanism of Recombination and Holiday structure formation. Enzymes involved

5. Chi-sites-function

6. Examples of consequences of recombination and gene conversion: hin recombinase and Salmonella flagella,

Four Requirements for Recombination

1. 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?

2. 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?

3. Recombination enzymes-the machinery of the recombination complex.

4. 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.

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

• ?

Figure 10.1

Figure 10.3

Single strand invasion model

Figure 10.4

Double strand break model

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

Figure 10.5

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 2565-base: 45 10246-base: 46 40968-base: 48 65536

Frequency of Chi-sites in the E.coli chromosome: 1 every 4-5kb

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

Figure 10.7

Figure 10.6

Figure 10.8

RuvABC complex-a large doughnut

Holiday Structure

1964 Robin Holiday

www.mun.ca/biochem/courses/3107/Lectures/Topics/Recombination_holliday.html

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

Importance of RecA for pathogens-Salmonella

Time-hours

Cfu

recA

Wild type

recA mutants cannot survive in host macrophages

Lethal Dose for miceWt: 10 cfurecA: 50,000+ cfu

What does this tell about the environment of the host?

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.