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Similar to GENETIC REQUIREMENTS FOR EFFICIENT REPLICATION THROUGH FISSION YEAST TELOMERES
Similar to GENETIC REQUIREMENTS FOR EFFICIENT REPLICATION THROUGH FISSION YEAST TELOMERES (20)
GENETIC REQUIREMENTS FOR EFFICIENT REPLICATION THROUGH FISSION YEAST TELOMERES
- 1. INVESTIGATING THE GENETIC REQUIREMENTS FOR EFFICIENT REPLICATION THROUGH FISSION YEAST TELOMERES Craig Gazzard
- 2. Telomere Structure • Heterochromatic structures that cap linear chromosomes • Made up of a repetitive sequence of nucleotides • Schizosaccharomyces pombe telomeres are comprised of a 300bp terminus region as well as 19kb of repetitive sub- telomeric elements • 5’-GGTTAC-3’ repetitive sequence
- 3. Replication of telomeres • Telomeres present a major obstacle to replication fork progression • Repetitive nature of the sequence results in secondary structure formation in the DNA • A wide array of proteins bind to telomeres as part of cell processes • Gradual shortening of telomere length • Telomerase nucleoprotein is recruited to lengthen the telomere
- 4. Replication of telomeres • Replication of human telomeres is a very complex process • Shelterin and CST (CTC1, STN1 and TEN1) complexes, Exonuclease 1 and telomerase • Bulk of replication occurs via semi-conservative replication • Telomerase compensates for gradual attrition of telomere G strand overhangs through addition of TTAGGG repeats • Extension of C strand is done by the polymerase-primase complex • Other factors such as the CST complex, STN1 and TEN1 proteins have recently been found to be involved
- 5. Replication of telomeres • Two nucleases are believed to be involved in generation of the G strand overhang – Apollo/SNM1B and Exonuclease 1 • Apollo is a 5’-3’ nuclease implicated in the generation of 3’ single strand overhangs • Negatively regulated by POT1 when POT1 is bound to TTAGGG repeats Exonuclease 1 subsequently generates a large G strand overhang before it is negatively regulated by CST CST is recruited by POT1 to correct over-resection by Exonuclease 1
- 6. Replication fork arrest with RTS1 • RTS1 is a DNA element that has been shown to play an important role in mating type switching in S. pombe • The sequence itself is around 800bp long and contains a 60bp partially conserved motif that repeats in full 3 times. • It is possible to induce replication fork arrest using RTS1 • Lambert et al in 2010 used an RTS1 system that was regulated by rtf1 gene activation
- 7. Fork arrest at telomeres • Aim to assess if DNA replication fork stalling has a role in the regulation of telomerase in S. pombe • Previous studies have found that replication fork stalling can produce a very good substrate for telomerase recruitment • Has been shown that replication fork arrival at telomeres and telomerase recruitment are linked.
- 8. Fork arrest at telomeres
- 9. Fork arrest at telomeres
- 10. Progress and results • Replication fork arrest at telomeres • Have successfully integrated the full cassette into the yeast chromosome. • Struggled to confirm that recombination had been successful by PCR and Southern blot • Cut the original plasmids to produce the intended construct after recombination and transformed into these strains
- 12. Fork arrest at the Ura4 locus • Aim to utilise the knowledge that Taz1 is essential for replication fork progression through telomeric DNA. • We seek to study this in Taz1 null cells through blocking replication forks approaching from the telomere and centromere • Using the same inducible RTS1 sequence as before • Plan to integrate the RTS1 block and telomeric repeats either side of the Ura4 locus in S. pombe.
- 13. Fork arrest at the Ura4 locus
- 14. Progress and results • Have integrated the various constructs into the yeast genome • Confirmed the integration and orientation of the construct for all 4 variations • Preliminary data shows that the telomeric DNA is having a silencing effect on the Ura4 gene. • Currently working to delete the Clr4 gene to remove the silencing effect
- 15. Future work • Fork arrest at telomeres • Confirm integration of the modified cassette • Telomere length assay • Fork arrest at the Ura4 locus • Confirm deletion of Clr4 gene • Delete the Taz1 gene • Conduct experiments to determine rate of Ura4 gene loss
Editor's Notes
- Characterised by a 30-400 nucleotide 3’ single strand overhang of G-rich strand. Overhang can fold back and invade double stranded telomeric region forking the telomeric loop and displacement loop. T-loop theorised to protect chromosome ends and prevent inappropriate activity of DNA repair mechanisms or telomerase.
- S. Pombe telomeres are bound by a wide range of proteins that have been found to be very similar to the proteins found in human telomeres. One key protein in yeast telomeres is Taz1 which has been found to be essential for replication through telomeres, this in turn recruits other factors like Rif1 and Rap1 which have secondary roles in telomeric regulation. Taz1 has been shown to be essential as replication forks that are deficient in taz1 have their progress through the telomeres hindered significantly, taz1 has also been found to play a major role in replication timing control by Tazumi et al 2012 and Dehe et al 2012 found that Taz1 is the dominant factor enforcing cell cycle regulation of telomere processing. TAZ1 NULL CELLS HAVE OVERACTIVE TELOMERASE = LONG TELOMERES. Pot1, Tpz1, Poz1, Ccq1 other proteins bind to telomeres Due to nature of replication with a leading and lagging strand with the lagging strand being synthesised discontinuously through the use of short RNA primers to allow polymerase to fill in the gaps with DNA nucleotides. The final RNA primer has been found to be positioned 70-100 nucleotides from the end of the telomere. Therefore the lagging daughter strand has an almost mature overhang size after replication which is subsequently blunt ended and an overhang is produced through post-replication resection of the C-rich strand. Leads to telomere shortening. Telomerase recruited to short telomeres to compensate for the gradual shortening of telomeres by semi conservative replication. Does this through reverse transcription of associated RNA component Terc to lenghten the 3’ strand (G-rich) while the polymerase primase fills in the C-rich strand
- Bulk by semi conservative – degradation as final primer positioned 70-100 bases away from the end Telomerase, when activated inserts a series of TTAGGG repeats onto the end of the telomere Polymerase-primase extends out the C-strand by copying the template placed by Telomerase Produces a blunt or near blunt telomere with no overhang
- 2 nucleases involved in generation of overhang – apollo and exo1 Apollo is 5’-3’ and is usually only used at telomeres to produce a short G strand overhang with a TTAGGG repeat for POT1 to bind to POT1 then inactivates Apollo and Exo1 is used to create a large overhang before CST mediated inactivation Prevents over-resection and allows cell to fill in strand that has been resected too far. CST is recruited by POT1 binding to correct overhang length Gives POT1 two crucial roles – prevention of Apollo hyper-resection and recruiting CST to correct lengths of overhangs produced by Exo1
- RTS1 found to have an important role in mating type switching. Mating type switching relies on the chemical modification of one strand of DNA at the mat1 locus, achieved by RTS1 terminating replication forks that are entering the mat1 locus from the wrong side that could interfere with switching. Has been used in previous experiments to block replication forks to allow further study and observation of how replication forks restart after being blocked. We plan to use a thiamine inducible system in our experiments. Through regulation of rtf1 by the thiamine-repressible promoter nmt41 – addition of thiamine will activate the RTS1 block.
- Using same RTS1 sequence as used by Lambert et al we will block replication forks approaching the telomeres and observe what happens to the telomere length. It may demonstrate that replication fork stalling triggers telomerase activation as it is known that replication forks struggle through telomeres and have a high risk of stalling and dissociating from the telomere – telomerase would fix any telomere length loss. Also may show how close to the telomere the replication fork has to be when telomerase is recruited, if it is quite distant then there must be a mechanism to promote this pre-mature association of telomerase, if it is quite close then it would suggest that there may be some telomere localised signal that promotes telomerase recruitment.
- General structure showing location of integrated cassette with regards to telomere and TAS elements. Shows original plan to integrate cassette then trigger recombination through FOA to produce the final construct.
- The INDUCIBLE RTS1 block will prevent replication forks from replicating the telomere. This system uses the Urg1 promoter which produces a rapid response to thiamine addition to induce the block. This was planned to be done in the recombined strains that have lost the Ura4 selective marker, and the FOA induced recombination event was crucial for this to work correctly, didn’t occur correctly (As shown by Southern blot in progress slides)
- Taz1 essential for progression through telomeric DNA, using Taz1 null strains allows us to use telomeric DNA as a block to the replication fork. Integrating this on the centromere proximal side of Ura4 allows us to block replication from the centromere, while RTS1 can be used on the telomere proximal side to do the same. ALL COMBINATIONS OF ORIENTATION USED IN THIS!!!! Expect to observe a high rate of Ura4 loss through plating on FOA with more growth from strains that have lost the Ura4 locus and vice versa after inducing the RTS1 block.
- Constructs that should block replication forks approaching from the telomere through RTS1 while only top strain will hinder replication through the telomeric sequence for forks approaching from the centromere. In these we would expect loss of the Ura4 locus in the top strain when the RTS1 block is induced as the replication fork will be blocked from both directions. Observations can be made by levels of growth on FOA plates with higher levels of growth indicating a higher level of Ura4 loss which will be caused by the blockage of both replication forks.