The University of Sheffield
Department of Molecular Biology and Biotechnology

Fungal Genetics


Dr A S H Goldman
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The interests of this laboratory are centred on the workings of meiosis, the cell division that leads to the production of gametes. The parent cell undergoing meiosis is diploid, containing one pair of each chromosome type. The daughter cells of meiosis are haploid, containing a single copy of each chromosome. In order to separate pairs of homologous chromosomes equally during the first meiotic division, it is necessary for homologous chromosomes to locate each other and lie side by side and eventually synapse. Concomitant with chromosome pairing high levels of genetic recombination are induced. The initiating event of meiotic recombination is formation of DNA double-strand breaks (DSBs) made by the protein Spo11. About half of the recombination events are reciprocal leading to crossovers. Crossovers are essential to most meiotic systems creating a tie between homologues before they separate at anaphase. The work in this laboratory is based on understanding how homologous chromosomes pair and the regulation of DSB repair (recombination) in meiosis.

 

All experimental work is done in the model organism Saccharomyces cerevisiae, which has proven to be extremely amenable to meiotic studies and is an excellent model for mammalian meiosis.

 

Localisation of telomeres to the nuclear periphery Figure 1: Localisation of telomeres to the nuclear periphery could explain their influence on accessibility of chromosomes domains for recombination. (A - E) The nuclear membrane in represented by the solid circle. The effective nuclear radius of 165 kb is based on the experimental finding that, when one insert is close to a telomere, minimum efficiencies of ectopic recombination are seen when the second insert is about 165 kb from its closest telomere. The small black squares represent insert loci on heterologous chromosomes which recombine, the concentric dotted circles represent shells within the nuclear space that are effectively limits to movement towards the center for the loci diagrammed next to them. The limit to movement towards the periphery, for any locus, is the nuclear envelope. A locus can occupy the space between these two limits. (F) The labels A to E correspond to the approximate levels of efficiency of ectopic recombination expected from the preceding scenarios and are overlaid on the entire recombination data set. Various situations are modeled that lead to changes in the sum of insert distances from the nearest telomere (SIDT), the relative mobility of different loci and the proportion of total nuclear volume that loci might occupy.
 
(A) When two inserts are close to a telomere, they have little ability to move in three-dimensions, but are in a relatively small volume and therefore recombine efficiently. ( B and C ) When one insert is close to a telomere and the other is interstitial, ectopic recombination would depend on the more telomere-distal insert being able to move to the region near the nuclear periphery where telomeric inserts are confined. As the distance between the telomere and the interstitial insert increases, the volume that could be occupied by the interstitial insert also increases, reducing the chance of interaction between the two sequences. ( D and E ) Similarly when both inserts are interstitial, moving them progressively further from a telomere increases the three-dimensional space in which they might reside. As a consequence the likelihood of ectopic contacts decreases.

 

Meiotic yeast cell  
Figure 2: Meiotic yeast cell with nuclear membrane illuminated by RFP and LacO chromosomal inserts illuminated by GFP. We are currently setting up an assay system that will allow us to survey the nuclear localisation of telomeres and other chromosome domains in various mutant backgrounds.

 

In molecular studies we have discovered that repair of DNA DSBs during meiotic recombination is partially dependent on the presence of wild type Spo11p, the protein that normally makes DSBs (3). We have constructed SPO11 and spo11-Y135f strains with a cutsite for the meiosis specific vde-endonuclease. The strains have been engineered so that repair of the vde-double strand break (VDE-DSB) can be via either inter-chromosomal gene conversion or intra-chromosomal single strand annealing (SSA). Genetic analysis in return to growth experiments reveals reduced gene conversion frequencies when only mutant Spo11p is available (Fig 3). Analysis of meiotic DNA supports this and indicates that processing of the DSB is modified and repair is more often by SSA in the mutant strain (Fig 4). Further analysis has shown that the rate and degree of single-stranded resection, which takes place during homologous recombination, is increased when SPO11 is absent.

 

Graph showing frequencies of gene conversion to ARG4+ in return to growth experiments. Figure 3: Graph showing frequencies of gene conversion to ARG4+ in return to growth experiments. After 8 hours sporulation, there is significantly more commitment to gene conversion in the strain expressing wild type Spo11p.

 

Southern analyses showing parental DNA, the vde-DSB and intra-chromosomal repair product (SSA product). Figure 4: Southern analyses showing parental DNA, the VDE-DSB and intra-chromosomal repair product (SSA product). Note in the strain with wild type Spo11 the DSB appears smeared as expected, but is discrete in the mutant. This could be due to faster processing into product in the mutant strain. The bar graphs represent quantified amounts of the SSA product. More of the vde cut sites are turned into SSA products in the mutant strain. Thus, that less VDE-DSB repair is by interchromosomal recombination when Spo11-DSBs are not present.

 

More recently we have examined the repair profile of VDE-DSBs in many mutant backgrounds that influence meiotic recombination. In particular we have used the VDE-DSB to examine repair in mutants that block repair of normal Spo11-induced DSBs. One aspect of great interest is to discover how recombination in meiosis is directed to be interchromosomal (making crossovers links between homologous chromosomes) rather than interchromosomal (more typical in mitosis). Overall, the data lead us to the important conclusion that the presence of Spo11-DSBs, and their normal processing, are major contributors to regulating the outcome of meiotic DSB repair in trans. While the presence of Spo11-DSBs is required to achieve interchromosomal repair, they are not sufficient. We suggest that Spo11-DSBs must be processed by downstream proteins for either activation or commitment to the interchromosomal repair route. One implication of these findings is that entering the meiotic cell cycle, per se, is not sufficient to activate the meiosis typical DSB repair pathway, but meiosis typical damage and DNA end processing must also occur.

 

Future studies will expand the molecular work on DNA to include biochemical work. In particular we are using western analysis and two-dimensional gels to look for protein modifications important for regulating meiotic recombination.

 

Selected Publications

1] Johnson, R,. V. Borde, M. J. Neale, A. Bishop–Bailey, M. North, S. Harris, A. Nicolas, and A. S. H. Goldman Excess single–stranded DNA inhibits meiotic double–strand break repair. (2007) (PLoS Genetics 3:e223).
Blundred R., K. Myers, T. Helleday, A. S. H. Goldman and Helen E. Bryant1* RECQL5 overcomes thymidine induced replication stress. (Submitted)
Terentyev, Y., R. Johnson, M. Neale, A. Bishop-Bailey, and A. S. H. Goldman. Evidence that MEK1 could positively enforce interhomologue double-strand break repair in addition its role in creating a barrier to sister chromatid repair. (In press Nucl. Acids Res.)
Lundin C., M. North, K. Erixon, K. Walters, D. Jenssen, A. S. H. Goldman*, T. Helleday* (2005) Methyl methanesulfonate (MMS) produces heat-labile DNA damage but no detectable in vivo DNA double-strand breaks. Nucleic Acids Res. 2005 Jul 11;33(12):3799-811. *[Joint senior author].
[3] Schlecht, H. S., M. Lichten and A. S. H. Goldman, 2004 Compartmentalization of the yeast meiotic nucleus revealed by analysis of ectopic recombination. Genetics 168:1189-1203.
Wiederkehr,C., R.Basavaraj, C.Sarrauste de Menthiere, L.Hermida, R.Koch et al., 2004 GermOnline, a cross-species community knowledgebase on germ cell differentiation. Nucleic Acids Res 32: D560-567.
Neale M. J., M.Ramachandran, H.Scherthan, E.Trelles-Sticken, and A.S.H.Goldman (2002). In Saccharomyces cerevisiae, wild-type levels of Spo11-induced double strand breaks are required for normal regulation of single strand resection during meiosis. Mol. Cell 9:835-846.
Borde V., A.S.H.Goldman and M.Lichten (2000). Direct coupling between meiotic DNA replication and recombination initIation. Science 290:806-809.
Goldman A. S. H. and M. Lichten (2000). Restriction of ectopic recombination by interhomolog interactions during Saccharomyces cerevisiae meiosis. Proc. Natl. Acad. Sci. (USA) 97:9537-9542.
*Armstrong S. J., *A. S. H. Goldman, R. M. Speed and M. A. Hultén (2000). FISH analysis of chiasma formation and first meiotic segregation in a human male translocation carrier t(11;22)(q23;q11): Regular quadrivalent formation with increased chiasma frequency and no evidence for preferential to 3:1 segregation. Am. J. Hum. Genet. 67:601-609. * [Joint first author].