Fungal Genetics

Professor A S H Goldman - HoD

Sheffield 222 2779
A.Goldman@sheffield.ac.uk

Career History

The interests of this laboratory are centered on the workings of meiosis, the cell division that leads to the production of gametes.

Figure 1: Prior to meiosis there is a round of DNA replication when (A) the chromosomes are relatively diffuse. (B) After replication each chromosome consists of two sister chromatids. Homologous (maternal and paternal) chromosomes pair up. In most organisms the process of recombination initiates during early chromosome pairing and is completed at the end of synapsis. (C) Homologues congress on the equator of the nucleus. They remain attached to each other due to the presence of crossovers and sister chromatids being held tightly together. (D) After attachment to the spindles the recombinant homologous chromosomes are separated during Anaphase I. (E) A second spindle is built and (F) sister chromatids segregate during Anaphase II, yielding (G) four unique haploid gametes or spores.

Through research into meiosis we aim to understand a phase of the eukaryotic life cycle that is key to providing the genetic variation on which natural selection and evolution depend.

Speciation also relies on the barriers in meiosis that prevent hybrid fertility. While this aspect of meiosis has been key to the evolution of so many Eukaryotic species, it also prevents the transfer of genes between species, something which crop breeders need to achieve to improve the quality or hardiness of crop species. So the ability to control meiosis could bring with it the promise of increased ability to modify and improve crops by breeding in desirable genes from related wild species.

Figure 2: Meiotic nuclei from yeast,Saccharomyces cerevisiae, expressing Zip1-GFP.For parental chromosomes can be separated during meiosis they must first pair up. After this they are tightly bound by the synaptonemal complex, which contain the protein Zip1. (A) During chromosome pairing and prior to synapsis Zip1 is seen in punctate structure. (B) When sysnapsis is complete Ziip1-GFP highlight each individual bivalent along its entire length.

Understanding meiosis can tell us much more than about fertility. Because meiosis includes a programme of induced DNA double-strand breaks and repair by homologous recombination, it provides an excellent model system for discovering the complex DNA transaction that are also important for genome stability in general. A loss of genome stability in turn is a major underlying cause of cancer and other developmental disorders such as microcephaly and neurodegenerative disease.

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 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. Most of the work in this laboratory is based on understanding how homologous chromosomes pair and the regulation of DSB repair (recombination) in meiosis.

Figure 3: Recombination in meiosis is stimulated by DNA double-strand breaks made by (A) the protein Spo11, which covalently binds to the DNA and is removed attached to an oligonucleotide. This starts off a process (B) called resection that requires Mre11 and Exo1; and creates 3´ single-stranded DNA. We model this process using (C) site specific double-strand breaks created by the meiosis specific self homing endonuclease VDE.

Figure 4: Much of our work is based on measuring products and single-stranded intermediates using reporter constructs in which there is a single VDE-induced double-strand break. (A) One reporter cassette allows us to see long resection tract repair using single-strand annealing versus gene conversion. (B) The second reporter cassette allows to compare the proportion of single-strand annealing products made from proximal or distal flanking repeated sequences. See references 3, 4, 6 and 10.

Figure 5: We have also used qPCR to the proportion of molecules in the population with different lengths of single-stranded DNA at both VDE- and Spo11-induced double-strand breaks. See references 2 and 3.

Most recently we have developed a computer model to describe the layout of chromosomes in the meiotic nucleus in 4-dimensions. This model serves the purpose of testing the theoretical significance of a specialised chromosome layout called the Bouquet.

Figure 6: Chromosomes reorganise early in meiosis to form the bouquet structure. (A) During mitosis the nucleus divides and centromeres move towards the microtubule organiser (spindle pole body in S. cereveisiae). (B) The centromeres remain clustered and close to the nuclear envelope in the Rabl configuration. (C) During early meiosis the telomeres become attached via SUN/KASH proteins (not shown) to the nuclear envelope and then (D) cluster near the microtubule organising centre in the bouquet configuration.

Our model supports the view that the bouquet may be sufficient to bring short chromosomes together, but the contribution to long chromosomes is less. We also found that persistence length is critical to how much influence the bouquet structure could have, both on pairing of homologues and avoiding contacts with heterologues.

Figure 7: The output of the computer model expressed as distance between alleles on homologous chromosomes (proportion of nuclear diameter; X-access) from one end of (A) the shortest and (B) the longest chromosome of S. cerevisiae. The distances between allelic loci are much shorter over the entire length of chromosome I, compared to chromosome IV, for which most of the chromosome is as far apart as predicted when telomeres are free form the nuclear periphery. See reference 1.

References

1. Penfold, C., Brown, P.E., Lawrence, N. and Goldman, A.S.H. (2012) Modeling Meiotic Chromosomes Indicates a Size Dependent Contribution of Telomere Clustering and Chromosome Rigidity to Homologue Juxtaposition. PLoS Comput. Biol. 8(5):e1002496.

2. Keelagher, R.E., Cotton, V.E., Goldman, A.S.H. and Borts, R.H. (2011) Separable roles for Exonuclease I in meiotic DNA double-strand break repair. DNA Repair (Amst), 10, 126-137.

3. Hodgson, A., Terentyev, Y., Johnson, R.A., Bishop-Bailey, A., Angevin, T., Croucher, A. and Goldman, A.S.H. (2011) Mre11 and Exo1 contribute to the initiation and processivity of resection at meiotic double-strand breaks made independently of Spo11. DNA Repair (Amst), 10, 138-148.

4. Terentyev, Y., Johnson, R., Neale, M.J., Khisroon, M., Bishop-Bailey, A. and Goldman, A.S.H. (2010) Evidence that MEK1 positively promotes interhomologue double-strand break repair. Nucleic Acids Res, 38, 4349-4360.

5. Blundred, R., Myers, K., Helleday, T., Goldman, A.S.H. and Bryant, H.E. (2010) Human RECQL5 overcomes thymidine-induced replication stress. DNA Repair (Amst), 9, 964-975.

6. Johnson, R., Borde, V., Neale, M.J., Bishop-Bailey, A., North, M., Harris, S., Nicolas, A. and Goldman, A.S.H. (2007) Excess single-stranded DNA inhibits meiotic double-strand break repair. PLoS Genet, 3, e223.

7. Lundin, C., North, M., Erixon, K., Walters, K., Jenssen, D., Goldman, A.S.H. and Helleday, T. (2005) Methyl methanesulfonate (MMS) produces heat-labile DNA damage but no detectable in vivo DNA double-strand breaks. Nucleic Acids Res, 33, 3799-3811.

8. Wiederkehr, C., Basavaraj, R., Sarrauste de Menthiere, C., Hermida, L., Koch, R., Schlecht, U., Amon, A., Brachat, S., Breitenbach, M., Briza, P. et al. (2004) GermOnline, a cross-species community knowledgebase on germ cell differentiation. Nucleic Acids Res, 32, D560-567.

9. Schlecht, H.B., Lichten, M. and Goldman, A.S.H. (2004) Compartmentalization of the yeast meiotic nucleus revealed by analysis of ectopic recombination. Genetics, 168, 1189-1203.

10. Neale, M.J., Ramachandran, M., Trelles-Sticken, E., Scherthan, H. and Goldman, A.S.H. (2002) Wild-type levels of Spo11-induced DSBs are required for normal single-strand resection during meiosis. Mol Cell, 9, 835-846.

11. Goldman, A.S.H. and Lichten, M. (2000) Restriction of ectopic recombination by interhomolog interactions during Saccharomyces cerevisiae meiosis. Proc Natl Acad Sci U S A, 97, 9537-9542.

12. Borde, V., Goldman, A.S.H. and Lichten, M. (2000) Direct coupling between meiotic DNA replication and recombination initiation. Science, 290, 806-809.

13. Armstrong, S.J., Goldman, A.S.H., Speed, R.M. and Hultén, M.A. (2000) Meiotic studies of a human male carrier of the common translocation, t(11;22), suggests postzygotic selection rather than preferential 3:1 MI segregation as the cause of liveborn offspring with an unbalanced translocation. Am J Hum Genet, 67, 601-609.