Page 48 - Genetics_From_Genes_to_Genomes_6th_FULL_Part2

6.5 Homologous Recombination at the DNA Level 207

The molecular intermediate formed at the conclusion The cell’s enzymatic machinery works to repair the
of Step 3 on Fig. 6.27 may have two alternative fates. damaged DNA site, and recombination is a side effect of
One pathway, depicted in Steps 4 through 6, results in this process.
crossing-over. The second pathway, shown in Steps 4′ and 5′,
does not yield a crossover, but one of the resultant chroma-
tids has a heteroduplex region. A summary: Evidence for the current molecular
model of homologous recombination
The double-strand-break repair model of meiotic recombi-
The crossover pathway nation was proposed in 1983, well before the direct obser-
The strand displaced by strand invasion in Step 3 now vation of any recombination intermediates. Scientists have
forms a second heteroduplex with the other 3′ single- now seen—at the molecular level—the formation of
stranded tail (Fig. 6.27, Step 4). DNA synthesis to extend double-strand breaks, the resection of those breaks to pro-
the two 3′ tails replaces the DNA that was degraded by the duce 3′ single-strand tails, and double Holliday junction
exonuclease, and DNA ligase reseals the DNA backbones structures. The double-strand-break repair model has become
(Fig. 6.27, Step 4). The result is that the two nonsister established because it explains much of the data obtained
chromatids are interlocked at two Holliday junctions from genetic and molecular studies as well as the six prop-
(Fig. 6.27, Step 5). The Holliday junctions move away from erties of recombination deduced from genetic experiments:
each other and thereby enlarge the heteroduplex between
them—a process called branch migration (Fig. 6.27, Step 5). 1. Homologs physically break, exchange parts, and re-
Now, the two nonsister chromatids must be separated. join. The Meselson-Weigle experiment with phage
The two chromatids disengage by the cutting and joining of lambda provided key evidence for this key aspect of
two strands of DNA at each Holliday junction. As shown in recombination (review Fig. 6.26).
Fig. 6.27 (Step 6), crossing-over (and recombination of 2. Crossing-over occurs between nonsister chromatids
flanking alleles) results when a different pair of DNA after DNA replication. When yeast dihybrid for linked
strands is cut and rejoined by resolvase and ligase enzymes genes sporulate, the appearance of T tetrads and the
at each junction. Because resolvase almost always cuts all rarity of NPDs make sense only if recombination hap-
four DNA strands, resolution of the double Holliday junc- pens at the four-strand, as opposed to the two-strand,
tions usually results in crossing-over. stage (review Fig. 6.25).
3. Breakage and repair generate reciprocal products of
recombination. Yeast and Neurospora tetrads are al-
The noncrossover pathway most always NPD, PD, or T because the reciprocal re-
Recombination initiated by Spo11 can also result in no combinants are found in the same ascus.
crossing-over through the action of an enzyme called 4. Recombination events can occur anywhere along the
anticrossover helicase. The helicase helps disentangle the DNA molecule. If enough progeny are counted, crossing-
invading strand from the nonsister chromatid, thus inter- over can be observed between any pair of genes in a
rupting Holliday junction formation (Fig. 6.27, Step 4′). variety of different experimental organisms.
Note that although the end result of this pathway is no 5. Precision in the exchange—no gain or loss of nucleo-
crossing-over (Step 5′), one of the resultant chromatids tide pairs—prevents mutations from occurring during
nonetheless contains a heteroduplex region. the process. Geneticists originally deduced the preci-
sion of crossing-over from observing that recombina-
Controlling where and when recombination occurs tion usually does not cause mutations; today, we know
this to be true from DNA sequence analysis.
Only cells undergoing meiosis express the Spo11 protein, 6. Gene conversion—the physical change of one allele
which is responsible for a rate of meiotic recombination in a heterozygote into the other—sometimes occurs
several orders of magnitude higher than that found in mi- as a result of a recombination event. In the next sec-
totically dividing cells. In yeast and humans, where meiotic tion, you will see how gene conversion is explained
double-strand breaks have been mapped, it is clear that by the formation of heteroduplexes during recombi-
Spo11 has a preference for cleavage of some genomic se- nation events.
quences over others, resulting in hotspots for crossing-over
(recall Fig. 5.17).
Unlike meiotic cells, mitotic cells do not usually initi- DNA Repair of Heteroduplexes Can Result
ate recombination as part of the normal cell-cycle program; in Gene Conversion
instead, recombination in mitotic cells is a consequence of
environmental damage to the DNA. As you will see in Yeast tetrad analysis allows us to see, just as Mendel
Chapter 7, X-rays and ultraviolet light, for example, can predicted, that alleles segregate equally into gametes.
cause either double-strand breaks or single-strand nicks. Diploids heterozygous at a particular locus produce two