roles in fertility and the maintenance of genome integrity The Drosophila WRN Exonuclease Ralph S. Lasala 1,2, Lynne S. Cox 2, and Robert D. C. Saunders 1 1 Department of Life Sciences, The Open University, Milton Keynes 2 Department of Biochemistry, University of Oxford, Oxford September 2008

Werner Syndrome (WS)‏ A progeria syndrome, and a model for ageing short stature greying of hair cataracts skin ulcerations chromosomal instability Autosomal recessive mutation of WRN

Chromosomal Defects in Cells from WS Patients Chromosome rearrangements Aberrant 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 CG7670 e strong hypomorph CG7670 D229V - 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 mwh 1 - 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 the fly is heterozygous for mwh 1 – the wing blade cells are normal, with a single hair each

Mitotic Recombination in CG7670 e04496

Project Aims 1.Characterise the female sterility (maternal effect lethality) of CG7670 e04496 homozygotes 2.Test germ-line dependence of female sterility 3.Evaluate rDNA replication in CG7670 e04496 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 7 th or 8 th 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

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

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 CG7670 e04496 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