5. Unidirectional transposition is initiated by the recipient MAT locus

17.5 Unidirectional transposition is initiated by the recipient MAT locus


A switch in mating type is accomplished by a gene conversion in which the recipient site (MAT) acquires the sequence of the donor type (HML or HMR). Sites needed for transposition have been identified by mutations at MAT that prevent switching. The unidirectional nature of the process is indicated by lack of mutations in HML or HMR.


The mutations identify a site at the right boundary of Y at MAT that is crucial for the switching event. The nature of the boundary is shown by analyzing the locations of these point mutations relative to the site of switching (this is done by examining the results of rare switches that occur in spite of the mutation). Some mutations lie within the region that is replaced (and thus disappear from MAT after a switch), while others lie just outside the replaced region (and therefore continue to impede switching). So sequences both within and outside the replaced region are needed for the switching event.




Figure 17.9 HO endonuclease cleaves MAT just to the right of the Y region, generating sticky ends with a base overhang.

Switching is initiated by a double-strand break close to the Y-Z boundary that coincides with a site that is sensitive to attack by DNAase. (This is a common feature of chromosomal sites that are involved in initiating transcription or recombination.) It is recognized by an endonuclease coded by the HO locus. The HO endonuclease makes a staggered double-strand break just to the right of the Y boundary. Cleavage generates the single-stranded ends of 4 bases drawn in Figure 17.9. The nuclease does not attack mutant MAT loci that cannot switch. Deletion analysis shows that most or all of the sequence of 24 bp surrounding the Y junction is required for cleavage in vitro. The recognition site is relatively large for a nuclease. Probably the recognition sequence occurs only at the three mating-type cassettes (Strathern et al., 1982).


Only the MAT locus and not the HML or HMR loci are targets for the endonuclease. It seems plausible that the same mechanisms that keep the silent cassettes from being transcribed also keep them inaccessible to the HO endonuclease. This inaccessibility ensures that switching is unidirectional.




Figure 17.10 Cassette substitution is initiated by a double-strand break in the recipient (MAT) locus, and may involve pairing on either side of the Y region with the donor (HMR or HML) locus.


Figure 14.5 Recombination is initiated by a double-strand break, followed by formation of single-stranded 3 F ends, one of which migrates to a homologous duplex.

The reaction triggered by the cleavage is illustrated schematically in Figure 17.10 in terms of the general reaction between donor and recipient regions. In terms of the interactions of individual strands of DNA, it follows the scheme for recombination via a double-strand break drawn in Figure 14.5; and the stages following the initial cut require the enzymes involved in general recombination. Mutations in some of these genes prevent switching.


Suppose that the free end of MAT invades either the HML or HMR locus and pairs with the region of homology on the right side. The Y region of MAT is degraded until a region with homology on the left side is exposed. At this point, MAT is paired with HML or HMR at both the left side and the right side. The Y region of HML or HMR is copied to replace the region lost from MAT (which might extend beyond the limits of Y itself). The paired loci separate. (The order of events could be different.)


Like the double-strand break model for recombination, the process is initiated by MAT, the locus that is to be replaced. In this sense, the description of HML and HMR as donor loci refers to their ultimate role, but not to the mechanism of the process. Like replicative transposition, the donor site is unaffected, but a change in sequence occurs at the recipient; unlike transposition, the recipient locus suffers a substitution rather than addition of material.



Research
Strathern, J. N. et al. (1982). Homothallic switching of yeast mating type cassettes is initiated by a double-stranded cut in the MAT locus. Cell 31, 183-192.



Genes VII
Genes VII
ISBN: B000R0CSVM
EAN: N/A
Year: 2005
Pages: 382

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