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