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From CellBiology

Spo11 Protein

Introduction

Topoisomerase 1: structurally related to Spo11 protein

In sexually reproducing organisms, recombination during Meiosis plays an important role. In an evolutionary point of view, it is a primary source of genetic variation by allowing exchange of genetic material from its homologous chromosome, giving selective advantage over asexual reproduction. Recombination also plays a mechanical role during meiosis as reciprocal exchange between homologous chromosome forms a physical connection that allow homologous pair of chromosome to accurately align themselves in the meiotic spindle for first meiotic division. Unsurprisingly, owing to its significance, process of recombination takes much of the prophase of meiosis, and is regulated tightly by interaction with multiple proteins, which ensures fidelity of the whole process. A protein of particular interest in recombination is Spo11, which is known to produce double-strand breaks (DSBs) in the DNA, which is an important step in initiating the meiotic recombination [1]. Failure to initiate DSB required for recombination, the chromosomes segregates aberrantly resulting in aneuploid gametes [2].

Structure

Spo11 gene in humans is located in chromosome 20, and it is comprised of 396 amino acid sequence. To this date, there is no crystal structure of Spo11 protein, and much of the structural studies are deduced from observation of the subunit of archaeal topoisomerase VI (Top6A), which shows similarity in sequence and its mechanism of function [3].

There are two distinct structural regions:

  • 5Y-CAP motif: Similar to catabolite gene activator protein (CAP) DNA binding domain and contains catalytic tyrosine residue.
  • Toprim domain: Four-stranded parallel beta sheet sandwiched between two pair of alpha-helices. Accommodates Mg2+ ions but its function is not known.

Function

Illustration of DSB formation by Spo11 protein. Red: Spo11, Dark Blue: double stranded DNA, black: cleavage site by Mre11-Rad50-Xrs2 complex

Key role of Spo11 protein is to generate DSB between homologous chromosomes to initiate meiotic recombination. The meiotic recombination is best understood in yeast, which is a good model species for understanding eukaryotic function.

  • Firstly, an intact DNA duplex is cleaved by Spo11 protein, which occurs leptotene of meiosis 1. This reaction is mediated by nucleophilic attack of the DNA phosphodiester backbone by tyrosine side chain on Spo11. This forms a phosphodiester bond between Spo11 and 5’ terminal strand and generating free 3’ OH-terminus [4]. This experimental observation is further supported by the fact that point mutation of the tyrosine residue in Spo11 has failed to produce DSB [5]
  • The determination of particular sequence for DSBs is not known, however open chromatin structures and repeated sequence are common targets for Spo11 gene [6]. In addition, site-directed mutagenesis of Spo11 gene showed reduced cleavage specificity of the enzyme. This highlights the fact that Spo11 is also involved in directing specificity of its function [7].
  • The free 3’ OH-terminus produced by DSBs repairs with the homologue rather than the sister chromatid, as a template, and thereby successfully producing recombinant homologous chromosomes.
  • After the DSBs are formed, the Spo11 attached to 5’ end of the DNA is cleaved from the chain by Mre11-catalysed endonucleolytic cleavage, along with short oligonucleotide still covalently bound to Spo11 [4].

Abnormalities

Mutation of the SPO11 gene shows distinct phenotype changes that can be related back to its function. Below outlines some of consequence and its significance Spo11 Mutation [8].

  • Male germ line cells arrested their meiosis prior to pachytene with little or no synapsis between homologous chromosomes. The arrested meiotic germ line cells then undergo apoptosis and both male and female species are infertile.
  • DSB formation is absent and consequently nondisjunction is observed in first meiotic division, which highlights the need for Spo11 to initiate recombination to allow accurate homologous chromosome segregation in meiosis 1.
  • The Spo11 mutations exhibit other phenotypes that may not be derived by loss of DSB function. For example, mutant germ line cell accelerates cell cycle progression through meiotic prophase [4].

Protein-Protein Interactions with Spo11

Although Spo11 is the catalytic unit that is involved in forming DSB, its activity requires interaction with other DSB proteins. So far products of at least 9 different genes have been indentified that interacts with Spo11: RAD30, MRE11, XRS2, MEI4, MER2, REC102, REC104, REC114, and SKI8. Whilst some of them are meiosis specific, others are expressed in mitotic cycle as well [9]. Mutations in these associated protein leads to defects similar defects in Spo11, highlighting their importance in recombination.
Illustration of protein-protein interaction with Spo11 that modulate DSB

As to date relatively little is known about the functional interaction between Spo11 and these associated proteins, however key interaction is summarised below and in figure (2) [10].

  • Ski8: direct partner of Spo11 and upon interaction with Spo11, it relocalizes from cytoplasm to nucleus and associates with meiotic chromosomes. This in turn allows Spo11 to recruit Rec102 and Rec104 to interact with to chromosomes.
  • Mer2: It has a phosphorylation site that can be phosphorylated by the cyclin-Cdk complex. Phosphorylation of Mer2 residue modulates its interaction with Spo11 and other related proteins.
  • Rec102/ Rec104: interact physically and functionally as a single functional unit. Residues in Rec104 can be phosphorylated; however its functional significance is unknown.

Glossary

  • Recombination: process by which genetic material is broken and joined to other genetic material
  • Homologous Chromosome: non-identical chromosomes that both contain information for the same biological features
  • Meiotic Spindle: structure involved in separation of chromosomes during mitosis and meiosis
  • Prophase: early phase of meiosis and mitosis
  • Aneuploid: abnormal number of chromosome derived from errors in meiosis
  • leptotene: first phase of prophase of meiosis, where chromosomes condense into thin threads
  • Sister chromatid: identical copies of a chromosome connected by a centromere
  • Synapsis: paring of the two homologous chromosome that occurs in meiosis 1

References

  1. Nag, D. K., Pata, J. D., Sironi, M., Flood, D. R., & Hart, A. M. (2006). Both conserved and non-conserved regions of Spo11 are essential for meiotic recombination initiation in yeast. Molecular Genetics & Genomics, 276(4), 313-321
  2. Louisa, E. J., & Bortsa, R. H. (2003). Meiotic recombinationL too mcuh of a good thing? Current Biology, 13(24), R953-R955.
  3. 3. Nichols, M. D., DeAngelis, K., Keck, J. L., & Berger, J. M. (1999). Structure and function of an archaeal topoisomerase VI subunit with homology to the meiotic recombination factor Spo11. EMBO Journal, 18(21), 6177-6188.
  4. 4.0 4.1 4.2 Keeney, S., Giroux, C. N., & Kleckner, N. (1997). Meiosis-specific DNA double-strand breaks are catalyzed by Spo11, a member of a widely conserved protein family. Cell, 88, 375-384.
  5. A Bergerat, Gadelle, D., & Forterre, P. (1994). Purification of a DNA topoisomerase II from the hyperthermophilic archaeon Sulfolobus shibatae. A thermostable enzyme with both bacterial and eucaryal features. Journal of Biological Chemistry, 269(44), 27663-27669.
  6. Jankowski, C., Nasar, F., & Nag, D. K. (2000). Meiotic instability of CAG repeat tracts occurs by double-strand break repair in yeast. Proceedings of the National Academy of Sciences, 97, 2134-2139.
  7. Diaz, R. L., Alcid, A. D., Berger, J. M., & Keeney, S. (2002). Identification of residues in yeast Spo11p critical for meiotic DNA double-strand break formation. Molecular Cell Biology, 22, 1106-1115.
  8. Romanienko, P. J., & Camerini-Otero, R. D. (2000). The mouse Spo11 gene is required for meiotic chromosome synapsis. Molecular Cell, 6, 975-987.
  9. Pâques, F., & Haber, J. E. (1999). Multiple Pathways of Recombination Induced by Double-Strand Breaks in Saccharomyces cerevisiae. Microbiology and Molecular Biology Reviews, 63, 349-404.
  10. Keeney, S., & Neale, M. J. (2006). Initiation of meiotic recombination by formation of DNA double-strand breaks: mechanism and regulation. Biochemical Society Transactions, 34(pt 4), 523-525.

Homework Page

Lecture 4: Nucleus

1. I found interesting that endoplasmic reticulum was continuous with the outer membrane. Previously have thought that they are separate organelle within the cell.

2. Existence of chromosome territories and the fact that space in nucleus are occupied by individual chromosomes were interesting.

Lecture 5: Exocytosis

Concepts that I found Hard to understand:

There was nothing major that I found hard to understand, and I believe lecture was comprehensive in understanding the whole process of exocytosis. One question that I might bring up is, whether smooth endoplasmic reticulum has any role in exocytosis?

Lecture 7: Mitochondria

Type of cellular processes that require a lot of energy:

  • Active transport, which requires transport of molecules against their concentration gradient, for example: Na/K ATPase, which utilises ATP produced from mitochondria.
  • Transcription, translation requires utilisation of ATP as a nucleotide, or used as part of activation or deactivation of various transcription factors and ribosomes.
  • Mitosis and Meiosis

Lecture 8: Cell Junctions

  • N-CAM: Neural Cell Adhesion Molecule is a glycoprotein expressed on the surface of neurons, glia, skeletal muscle and natural killer cells. NCAM has been implicated as having a role in cell-cell adhesion, neurite outgrowth, synaptic plasticity, and learning and memory.
  • Ng-CAM: abbreviated for Neuron-Glia Cell Adhesion Molecule, and mediates neuron-neuron adhesion by a homophilic mechanism.
  • L-CAM: stands for Liver cell adhesion molecule, which play role in calcium dependent cell to cell adhesion in the liver.
  • I-CAM: stands for Intercellular Adhesion Molecule, and it mediates adhesive interactions important for antigen-specific immune response.

Lecture 10: Intermediate Filaments

What is the name of the epidermal layer between the basal and granulosa layer and how does it relate to intermediate filaments?

The intermediate layer between strata basale and strata granulosum is the strata spinosum. This a layer of skin cell that starts keratinization, which are intermediate filaments, forming filamentous polymers.

Lab 6: Cytoskeleton Laboratory

Q: f you've seen differences in the distribution of phenotypes in Tm4 over-expressing B35 cells versus control B35 cells, describe these differences. Formulate a hypothesis with regards to what changes on the molecular level may have occurred due to the over-expression of Tm4 that lead to morphological changes that you have observed

the phenotype of the Tm4 over-expressing versus control B35 cells:

  • Total for [A] Phenotype: A = 4 B = 14 C = 43 D = 61 E = 75 F = 4
  • Total for [B] Phenotype: A = 9 B = 66 C = 126 D = 74 E = 18 F = 27

Type A phenotype: shows more elongated type of cells, which are dominated by prolonged and stringed cells

Type B phenotype: shows more broken fan and stumped phenotype, and vastly increased cell population compared to phenotype A.

Lecture 14: Confocal Microscopy

What are the 2 main forms of generating confocal microscopy?

The two forms are laser and spinning disk

Lecture 15: Cell Cycle

What does S phase mean?

S phase is short for synthesis phase, where DNA replication occurs