roles in fertility and the maintenance of genome integrity the drosophila wrn exonuclease ralph s....
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roles in fertility and the maintenance of genome integrityThe Drosophila WRN Exonuclease
Ralph S. Lasala1,2, Lynne S. Cox2, and Robert D. C. Saunders1
1Department of Life Sciences, The Open University, Milton Keynes2Department of Biochemistry, University of Oxford, Oxford
September 2008
Werner Syndrome (WS)
A progeria syndrome, and a
model for ageing
short staturegreying of haircataractsskin ulcerationschromosomal instability
Autosomal recessive
mutation of WRN
Chromosomal Defects in Cells from WS Patients
Chromosome rearrangementsAberrant mitotic recombination
Sensitivity to DNA-damaging agents such as camptothecin
Difficulty in overcoming obstacles to replication
Asymmetry in normal bidirectional
progression of replication forks
RecQ homologues from a range of taxa are shown
Domain structure is indicated
Only WRN contains an exonuclease domain in addition to the helicase domain
WRN-like helicases have only been found in vertebrates
In other taxa helicase and exonuclease functions are thought to be on separate polypeptides
WRN homologues
Why Drosophila?
WS material limiting
Difficult to analyse combined helicase/exonuclease – thought to reside in separate polypeptides in Drosophila
Drosophila has powerful genetic and transgenic technology
CG7670 encodes a homologue of WRN exonuclease
Drosophila WRN Exonuclease (DmWRNexo)353 amino acids (~40kDa)
3'-5' exonuclease activity shown in vitro (Boubriak et al, submitted)
CG7670 mutants
• CG7670e04496 - strong hypomorph• CG7670D229V - weaker, point mutation
• two phenotypes:– increased mitotic recombination– hypersensitivity to camptothecin
• males are fertile• females are sterile (maternal effect lethal?)
this cell is homozygous for mwh1
- will give rise to a mwh clone
this cell is homozygous for the wild type allele of mwh
- will give rise to a wild type clone, indistinguishable from the rest of the wing blade cells
mwh
+
mwh
+ +
mwh
mwh
+ mwh
+
the fly is heterozygous for mwh1 – the wing blade cells are normal, with a single hair each
Mitotic Recombination in CG7670e04496
Project Aims
1. Characterise the female sterility (maternal effect lethality) of CG7670e04496 homozygotes
2. Test germ-line dependence of female sterility
3. Evaluate rDNA replication in CG7670e04496
homozygotes, both genetically, andusing molecular techniques
Early Embryogenesis
• a syncytium of nuclei
– 13 nuclear divisions following fertilisation
– rapid division cycles lasting 10 mins (S - M)
– no G1 or G2 phases
– after 7th or 8th division, some reach surface (pole cells form)
– during telophase of cycles 8 and 9, migrate to the cortex
• syncytial blastoderm (cycles 10-13)– zygotic transcription
• cellularisation
Foe and Alberts, 1983
Campos-Ortega and Hartenstein, 1985
No Defect
No Defect
Gaps
Asynchrony
Anaphase Bridge
Defects in CG7670 Mutant Embryos
• low levels of DmWRNexo--- defects in chromosome segregation and mitotic synchrony during early embryogenesis
• defective nuclei are removed--- patches or gaps
0
2
4
6
8
10
12
%
rDNA and bobbed
Ribosomal RNA genes in tandem arrays of ~200 copies
Located on X and Y chromosomes
Partial or complete loss of rDNA locus leads to bobbed phenotype
Does loss of DmWRNexo lead to difficulty in rDNA replication?
Assay for rDNA instability in CG7670e04496
X chromosomes propagated patroclinously in homozygous mutant background
Assay for appearance bobbed phenotype
PCR assay for subtler loss of rDNA
What Next
• immunostaining• more microscopy• live imaging• rDNA / bobbed
experiment– PCR-based assays
• detailed analysis of rDNA stability– DNA fibre spreading
Acknowledgements
Robert Saunders
Lynne Cox
David Clancy
Ivan Boubriak
Penelope Mason