Question 8.6. According to the Holliday model for genetic recombination, what factor determines the length of the heteroduplex in the recombination intermediate?
Question 8.7. Holliday junctions can be resolved in two different ways. What are the consequences of the strand choice used in resolution?
Question 8.8. Why do models for recombination include the generation of heteroduplexes in the products?
Question 8.9. Consider two DNA duplexes that undergo recombination by the double-strand break mechanism. The parental duplex indicated by thin lines has dominant alleles for genes M, N, O, P, and Q, and the parental duplex shown in thick lines has recessive alleles, indicated by the lower case letters. The recombination intermediate with two Holliday structures is also shown.
- a) What duplexes result from resolution of the left Holliday junction vertically and the right junction horizontally?
- b) After the vertical-horizontal resolution, what will the genotype be of the recombination products with respect to the flanking markers M and Q? In answering, use a slash to separate the designation for the 2 chromosomes, each of which is indicated by a line (i.e. the parental arrangement is M___Q / m___q).
- c) If the products of the vertical-horizontal resolution were separated by meiosis, and then replicated by mitosis to generate 8 spores in an ordered array (as in the Ascomycetefungi), what would be the phenotype of the spores with respect to alleles of gene O? Assume that the sister chromatids of these chromosomes did not undergo recombination in this region (i.e. one parental duplex from each homologous chromosome remains from the 4n stage).
For the next 3 problems, consider two DNA duplexes that undergo recombination by the double-strand break mechanism. The parental duplex denoted by thin black lines has dominant alleles (capital letters) for genes (or loci) K, L, and M, and the parental duplex denoted by thick gray lines has recessive alleles, indicated by k, l, m. The genes are shown as boxes with gray outlines. In the diagram on the right, the double strand break has been made in the L gene in the black duplex and expanded by the action of exonucleases.
Question 8.10. When recombination proceeds by the double-strand break mechanism, what is the structure of the intermediate with Holliday junctions, prior to branch migration? Please draw the structure, and distinguish between the DNA chains from the parental duplexes.
Question 8.11. If the recombination intermediates are resolved to generate a chromosome with the dominant K allele of the K gene and the recessive m allele of the M gene on the same chromosome (K___m), which allele (dominant L or recessive l) will be be at the L, or middle, gene?
Question 8.12. If the left Holliday junction slid leftward by branch migration all the way through the K gene (K allele on the black duplex, k allele on the gray duplex), what will the structure of the product be, prior to resolution?
Question 8.13. According to the original Holliday model and the double-strand break model for recombination, what are the predicted outcomes of recombination between a linear duplex chromosome and a (formerly) circular duplex carrying a gap in the region of homology? The homology is denoted by the boxes labeled ABC on the linear duplex and ac on the gapped circle. The regions flanking the homology (P and Q versus X and Y) are not homologous.
The results of an experiment like this are reported in Orr-Weaver, T. L., Szostak, J. W. and Rothstein, R. J. (1981) Yeast transformation: a model system for the study of recombination. Proc. Natl. Acad. Sci. USA 78: 6354-6358. These data were instrumental in formulating the double-strand-break model for recombination.
Question 8.14.A variety of in vitro assays have been developed for strand exchange catalyzed by RecA. For each of the substrates shown below, what are the expected products when incubated with RecA and ATP (and SSB to facilitate removal of secondary structures from single-stranded DNA)? In practice, the reactions proceed in stages and one can see intermediates, but answer in terms of the final products after the reaction has gone to completion.
In each case, the molecule with at least partical single stranded region is shown with thick blue strands, and the duplex that will be invaded is shown with thin red lines. The DNA substrates are as follows.
- A. Single-stranded circle and duplex linear. The two substrates are the same length and are homologous throughout.
- B. Single-stranded short linear fragments and duplex circle. The short fragments are homologous to the circle.
- C. Single-stranded linear and duplex linear. The two substrates are the same length and are homologous throughout.
- D. Gapped circle and duplex linear. The intact strand of the circle is the same length as the linear and is homologous throughout. The gapped strand of the circle is complementary to the intact strand, of course, but is just shorter.