Page 44 - Genetics_From_Genes_to_Genomes_6th_FULL_Part2

6.5 Homologous Recombination at the DNA Level 203

one long DNA molecule, a logical expectation is that they heavy A B chromosomes would recombine with light a b
should show some physical signs of this breakage and rejoin- chromosomes. If crossing-over occurred through breakage
ing at the molecular level. To evaluate this hypothesis, re- and reunion of double-stranded DNA molecules, then some
searchers chose a bacterial virus, lambda, as their model A b recombinant phages from the lysed cells should have a
organism. Lambda had a distinct experimental advantage for density almost as heavy as that of the parent phages that were
this particular study: It is about half DNA by weight, so the A B (Fig. 6.26). In contrast, few if any recombinants of geno-
density of the whole virus reflects the density of its DNA. type a B should be composed of mostly heavy DNA.
The experimental technique was similar in principle to Because the phages had replicated in light medium, re-
the one Meselson and Stahl used to monitor a change in DNA combinant phages could be found throughout the gradient.
density during DNA replication. In this case, however, the re- However, the key result was that a substantial proportion of
searchers (again Matthew Meselson but with a different col- the A b recombinants were indeed found near the heaviest
laborator, Jean Weigle) monitored DNA density to look at density along with A B parent molecules. This result makes
recombination. They used two strains of bacterial viruses that sense only if the A b chromosomes consisted mostly of
were genetically marked to keep track of recombination. They double-helical heavy DNA, as expected for the kind of
grew the wild-type strain (A B) in medium with heavy iso- chromosomal breakage and reunion shown in Fig. 6.26.
topes of carbon and nitrogen, and a strain with mutations in
two genes (a b) in medium with the normal light isotopes of
these atoms (Fig. 6.26). Meselson and Weigle then infected Crossing-Over at the Molecular
bacterial cells growing in normal (light isotope) medium with Level: A Model
so many phages of each type that every cell was infected with
both viral strains. After allowing time for the phages to repli- Biochemical experiments performed mostly in yeast have in-
cate, recombine, and repackage their DNA into virus particles, formed our present understanding of the molecular mecha-
the experimenters isolated the viruses released from the lysed nism for meiotic recombination. Researchers have found that
cells and analyzed them on a density gradient. the protein Spo11, which plays crucial roles in initiating mei-
It was important to the design of the experiment that both otic recombination in yeast, is homologous to a protein es-
genes A and B were close to one end of the viral chromosome sential for meiotic recombination in nematodes, plants, fruit
(Fig. 6.26). The idea was that some of the original phage flies, and mammals. This finding suggests that the mecha-
chromosomes would undergo recombination before replicat- nism of recombination presented in detail in Fig. 6.27—and
ing in the light isotope medium. For example, some of the known as the double-strand-break repair model—has been
conserved throughout the evolution of eukaryotes.
In the figure, we focus on two nonsister chromatids,
Figure 6.26 DNA molecules break and rejoin during even though recombination takes place at the four-strand
recombination. Meselson and Weigle infected E. coli cells with stage. Furthermore, we use the term recombination event to
two different genetically marked strains of bacteriophage lambda describe the molecular process initiated by Spo11, whether
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15
previously grown in the presence of heavy ( C and N) or light or not it results in crossing-over. As you are about to see,
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( C and N) isotopes. After growth on light medium, they spun the the molecular details of recombination events are such that
progeny bacteriophages on a CsCl density gradient. Some A b
genetic recombinants (but almost no a B recombinants) had a crossing-over (reciprocal exchange of double-stranded
heavy density almost the same as that of the A B parent. DNA of nonsister chromosomes) results from the
A B Spo11-mediated process only some of the time.
Heavy
a b
Light Initiating recombination
Recombination
between genomes A meiotic recombination event begins when Spo11 makes
that had not replicated a double-strand break in one of the four chromatids
during coinfection (Fig. 6.27, Step 1). Next, in a process called resection, an
A b
Heavy exonuclease (an enzyme that removes nucleotides from an
end of a DNA molecule) degrades one strand of DNA from
Light both sides of the cleavage, leaving 3′ single-stranded tails
a B
(Fig. 6.27, Step 2). In the next set of reactions, called
Parents strand invasion, one single-stranded tail displaces the cor-
Light a b A B Heavy responding strand on the nonsister chromatid (Fig. 6.27,
Step 3). Strand invasion results in the formation of a
a B A b
Recombinant heteroduplex region (from the Greek hetero meaning other
daughters or different) in which the DNA molecule is composed of one
(Number of phages)
strand from each nonsister chromatid (Fig. 6.27, Step 3).