Modeling Werner Syndrome
in Drosophila melanogaster
Hyper-recombination in Flies Lacking
WRN-like Exonuclease
LYNNE S. COX,a DAVID J. CLANCY,b IVAN BOUBRIAK,a
AND ROBERT D. C. SAUNDERSb
aDepartment of Biochemistry, University of Oxford, Oxford, United Kingdom
bDepartment of life Sciences, The Open University, Milton Keynes,
United Kingdom
ABSTRACT: Human progeroid Werner syndrome provides the current
best model for analysis of human aging, recapitulating many aspects of
normal aging as a result of mutation of the WRN gene. This gene en-
codes a RecQ-type helicase with additional exonuclease activity. While
biochemical studies in vitro have proven invaluable in determining sub-
strate specificities of the WRN exonuclease and helicase, it has been diffi-
cult to dissociate the two key enzyme activities in vivo. We are developing
Drosophila as a model system for analysis of WRN function; the suitabil-
ity of Drosophila for extensive and sophisticated genetic manipulation
permits us to investigate regulatory pathways and the impact of WRN
loss at organismal, cellular, and molecular levels. BLASTP screening of
the Drosophila genome with human WRN sequence allowed us to identify
three RecQ helicases with strong homology to human WRN, a presumed
helicase component of the spliceosome, and two DEAH-box putative RNA
helicases with weaker WRN homology. None of these helicases contain
a WRN-like exonuclease domain, but two potential WRN-like exonucle-
ases in flies encoded by the loci CG7670 and CG6744 were also identified
in the BLAST search. CG6744 and CG7670 are more closely related to
human WRN than to each other. We have obtained a fly strain with a
piggyBac insertional mutation within the CG6744 locus, which decreases
expression of the encoded mRNA. Such flies show elevated levels of so-
matic recombination. We suggest that WRN-like exonuclease activity is
critical in maintaining genomic integrity in flies.
KEYWORDS: Werner syndrome; WRN; DNA recombination; Drosophila;
exonuclease; CG6744; CG7670; RecQ helicase; 3′-5′ exonuclease
domain-like 2 protein; genome instability
Address for correspondence: Lynne S. Cox, Department of Biochemistry, University of Oxford,
South Parks Road, Oxford, OX1 3QU, UK. Voice: +44-1865-275721; fax: +44-1865-275721.
lynne.cox@bioch.ox.ac.uk
Ann. N.Y. Acad. Sci. 1119: 274–288 (2007). C© 2007 New York Academy of Sciences.
doi: 10.1196/annals.1404.009
274

COX et al. 275
INTRODUCTION
Werner syndrome (WS) is a human progeroid syndrome manifesting pre-
dominantly in early adulthood when patients show greatly accelerated signs
of normal aging, including graying of hair, type II diabetes, atherosclerosis,
and cardiovascular disease.1 They also have a very high incidence of sarcomas
which, with cardiovascular disease, are the leading causes of death of WS pa-
tients (reviewed in Ref. 2, 3). These pleiotropic effects result from mutation
of a single gene, WRN ,4 which encodes a protein of 1432 amino acids and
which possesses both helicase activity characteristic of the RecQ family5 and
3′-5′ exonuclease activity6,7 with strong similarities to the DnaQ family of
proofreading nucleases.8 Additionally, WRN shares RQC and HRDC domains
with other members of the RecQ family; these domains probably serve roles
in DNA binding and protein–protein interactions (reviewed in Ref. 9).
Loss of WRN in cultured cells results in very rapid onset of replicative
senescence after only 9 to 11 population doublings,10 while at the subcellular
level, a high degree of DNA recombination can be observed,11–13 together with
abnormalities in DNA replication14–16 and hypersensitivity to a subset of DNA-
damaging agents, including camptothecin17–19 (CPT) and 4-nitroquinoline ox-
ide.20,21 WRN has been postulated to be involved in many related aspects
of DNA metabolism, including DNA replication, recombination, repair, tran-
scription, and telomere maintenance (reviewed in Ref. 3). The importance of
WRN in restraining illegitimate recombination is such that WRN has been
termed a caretaker tumor suppressor.22 DNA recombination, replication, and
drug sensitivity phenotypes in WS cells can be overcome by ectopic expres-
sion of a Holliday junction resolvase,21,23 suggesting that an accumulation of
Holliday junctions in the absence of WS leads to the hyper-recombinant and
proliferative deficits characteristic of WS.
Analysis of recombinant or immunoprecipitated WRN protein has been
extremely useful in elucidating DNA template specificities for nuclease and
helicase activities and in exploring WRN protein interactions (reviewed in
Ref. 24). Cell biology studies have been largely confined to fibroblastic or
lymphoblastic lines derived from WS patients, which are by their nature ge-
netically heterogenous and further complicated by the hyper-recombinant phe-
notype of cells lacking WRN. To overcome such issues of differences in cell
background, isogenic cell lines in which WRN function has been ablated by
RNA interference (RNAi) treatment have been developed (e.g., Ref. 25), but
there appears to be strong selective pressure against stable RNAi knockdown
of WRN in many cell lines (Cox L.S., Faragher R.G.A., unpublished results).
Moreover, such treatment (when successful) eliminates all WRN function and
does not allow distinction between exonuclease and helicase activities. Whole
organism mouse models of WRN loss have been established; while lipodystro-
phy was seen on deletion of part of the WRN helicase domain,26 a premature
aging phenotype was only detectable on WRN loss if mice also were null for

276 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES
Terc,27 the RNA component of the enzyme responsible for telomere mainte-
nance. This may reflect significant differences in aging mechanisms between
rodents and humans. Late generation Terc−/− Wrn−/− mice do indeed show
many of the phenotypes observed in human WS27 and such null mice may
provide a good animal model for WS. However, mice are, in laboratory terms,
long-lived and genetic manipulation is difficult.
Dual function exonuclease–helicase WRN has only been described in ver-
tebrates. The paradigm RecQ from Escherichia coli possesses only helicase
activity and associates with a nuclease, RecJ, when acting in recombination.28
Similarly, WRN-like RecQ helicases in Arabidopsis thaliana,29–31 Caenorhab-
ditis elegans,32,33 and Neurospora crassa34 are thought to associate with a
cognate exonuclease to carry out their role in maintaining genome stability.
The RecQ homologue Rqh1 protein in fission yeast is important in suppress-
ing illegitimate recombination but appears not to require helicase activity to
do so,35,36 suggesting that its association with a nuclease may be critical in
this context. Moreover, WRN exonuclease has been implicated in maintaining
genome stability after irradiation.37 BLM helicase, which can in vitro resolve
double Holliday junctions by dissolution without the need for exonucleases,38
may, in cells, associate with a nuclease, such as mus81, in suppressing ille-
gitimate recombination.39 Dissection of the roles and importance of nuclease
and helicase activities of WRN has been attempted by use of point mutations
in the exonuclease domain (e.g., D82A, E84A) or the helicase domain (e.g.,
K577M). Although such studies have shown utility in dissecting some aspects
of WRN’s roles (e.g., in survival versus recombination40), such mutant proteins
may possess dominant negative activity,41 preventing a complete investigation
of the distinct enzymatic activities in cells. To overcome this, it would be useful
to study WRN action in a whole-organism metazoan model in which the two
enzymatic activities are encoded as separate polypeptides. Arabidopsis studies
have proven very useful in highlighting similarities across phylogenetic king-
doms,29–31 suggesting that WRN function can be reconstituted by the action
of two distinct polypeptides with exonuclease and helicase activities, as in
E. coli.28 Such studies cannot, however, inform on the impact of WRN loss
at the level of animal development, tissue organization, and life span. While
C. elegans has the advantage of a very short life span and excellent genetics,
including simple RNAi manipulation, the developmental programming result-
ing in determination of the fate of every cell in the adult organism does not
allow analysis of stochastic elements that are thought to be so important in
human aging. We therefore turned to the fruit fly Drosophila melanogaster for
which sophisticated techniques of genetic manipulation and highly annotated
genome sequence42 are available. Furthermore, Drosophila is relatively short-
lived and undergoes distinct developmental stages with mitotic and postmitotic
aspects akin to aspects of development in more complex organisms.
In order to use Drosophila as a model for WS, however, it was necessary
to identify candidate WRN-like genes. Here, we briefly describe the family of

COX et al. 277
RecQ-like proteins in the fly, then look in more detail at a WRN candidate
nuclease, CG6744. We examine the phenotype of flies carrying a piggyBac
insertional mutation in the CG6744 locus which we show leads to a decrease
in CG6744 mRNA levels. We find a moderately elevated rate of recombina-
tion in these mutant flies, indicative of a role for WRN-like exonucleases in
maintaining genome stability.
RESULTS AND DISCUSSION
Identification of WRN-like Proteins in Drosophila Melanogaster
A BLAST search43 was performed against all known and predicted D.
melanogaster proteins using the human WRN protein sequence. TABLE 1 shows
the proteins identified with the strongest similarities to human WRN. Of these
proteins, DmBLM44 (encoded by the mus309 locus) has the greatest similarity
to human WRN, with Drosophila RecQ545 and RecQ4 showing significant
regions of homology within the helicase domain (TABLE 1 top, see also Ref.
46). The assignment of mus309 to the BLM rather than the WRN branch of
the RecQ helicases is based on a higher percentage amino acid identity and
similarity with full-length human BLM than WRN,44 and the absence of an
exonuclease domain characteristic of WRN. Within the helicase domain, how-
ever, the product of the mus309 locus is remarkably similar to human WRN
(data not shown). It is conceivable that the protein encoded by the mus309
locus may act as a helicase with both BLM and WRN-like capacities in flies.
Three additional helicases encoded by CG6418, CG6227, and CG10077
show limited homology with human WRN (TABLE 1A), predominantly in the
DEAH-box typical of RNA helicases. None of the three Dm RecQ homo-
logues, nor the related presumptive RNA helicases, showed any regions that
are likely to function as a nuclease, as judged from sequence similarity (data not
shown). However, the BLAST screen also identified two candidates, CG7670
and CG6744, with WRN-like exonuclease domains (TABLE 1 bottom). While
CG6744 has slightly greater identity to human WRN (34% compared with 33%
for CG7670), it is less similar overall, and this is reflected by the significant
difference in E-values (TABLE 2).
Sequence alignments were then performed with predicted proteins encoded
by CG6744, CG7670, and human WRN protein.47 Both CG7670 and CG6744
proteins possess conserved residues known to be critical for nuclease activity
in human WRN (FIG. 1A); in particular, acidic amino acids D83 and E85 in
CG6744, equivalent to human WRN D82 and E84, respectively, that have
been demonstrated by crystallographic analysis to be involved in coordination
of two Mg2+/Mn2+ ions essential for nuclease catalysis.8 In addition, human
WRN aspartate 143 indirectly interacts with the outer of the two metal ions via

278 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES
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COX et al. 279
TABLE 2. Homologies of WRN-like exonucleases from Drosophila melanogaster and other
invertebrates with human WRN
Protein pairs Identities Similarities E-value
CG6744: CG7670 26% (47/177) 41% (73/177) 3e−6
CG6744: hWRN 34% (51/150) 46% (70/150) 5e−12
CG7670: hWRN 33% (72/212) 52% (112/212) 1e−22
CG6744: AtWEX (At4g13870) 35% (55/154) 51% (79/154) 3e−13
CG6744: Mut7 (C. elegans) 35% (21/59) 57% (34/59) 0.032
CG6744: hEXDL2 (human) 40% (212/520) 59% (311/520) 2e−101a
BLASTP searches were performed using NCBI BLAST.
aNCBI BLAST uses the full length EXDL2 protein sequence, while SwissProt gives an E-value of
4e−94 due to omission of the N terminal portion of EXDL2.
two water molecules8; interestingly, this residue is also conserved in CG6744
(D142). FIGURE 1A also shows the relationship between CG6744, CG7670,
and WRN-like exonucleases from other invertebrates (Arabidopsis At4g13870
and C. elegans Mut7), further supporting their identification as fly orthologs
of WRN-like exonucleases.
We then assessed similarities and identities for pairwise comparisons
(TABLE 2). CG6744 and CG7670 are related to each other, sharing key fea-
tures in the putative 3′-5′ exonuclease domain, but the E-value of 3e−6 is
much less significant than that for either WRN-CG7670 or WRN-CG6744.
Note that these E-values were obtained from BLAST scores within the ex-
onuclease domain only. From these scores, it is likely that CG7670 is the fly
ortholog of WRN; we describe the phenotypes associated with CG7670 muta-
tion in detail elsewhere.47 However, in T-Coffee alignments, allowing for large
gaps in the sequence, we found that CG6744 shared significant regions of ho-
mology with human WRN within its helicase ATP-binding domain, the RQC
region, and also C-terminal of these regions prior to the HRDC domain (data
not shown). Interpretation of this finding was further complicated by the fact
that in addition to its homology to WRN, CG6744 also shows 40% identity
and 59% similarity with the putative human exonuclease 3′-5′ domain-like 2
protein (GenPept accession number NP 060669) (TABLE 2 and FIG. 1B). The
E-value of 2e−101 strongly suggests that CG6744 represents the ortholog of
this so far uncharacterized human 3′-5′ exonuclease domain-like 2 protein,
presumed, on the basis of sequence homology to E. coli pol I, to be involved
in proofreading or a similar single-strand nuclease activity.
To obtain a phylogenetic tree showing the relationship between CG6744
and other WRN-like 3′5′ exonucleases from a range of species (FIG. 2A), we
used the sequence of CG6744 protein to carry out BLAST searches of the
entire nonredundant protein database. Such plots show relationships between
the most closely related proteins from a variety of phyla and species; because
of the much higher degree of similarity between CG6744 and EXDL2 than

280 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES
FIGURE 1. WRN-like exonucleases from Drosophila melanogaster. (A) Alignments of
Drosophila WRN-like exonucleases CG7670 and CG6744 with human WRN and WRN-like
exonucleases AtWEX from Arabidopsis (At4g13870) and Mut7 from C. elegans (CeMut7).
Asterisks indicate acidic residues critical for nuclease activity. (B) Alignment of CG6744
with human 3′-5′ exonuclease domain-like 2 protein, EXDL2. In both parts, amino acid
identities are shown in black, while different levels of similarity are depicted by varying
levels of gray shading.
between CG6744 and human WRN (TABLE 2), this plot does not include human
WRN. To clarify the relatedness of CG6744 with nucleases from other phyla,
including Arthropoda, we selected key examples from a range of phyla to
derive a radial plot of relationships. From this diagram (FIG. 2B), it is clear
that CG6744 is most similar to 3′-5′ exonucleases from other arthropods (e.g.,
Anopheles) but that it also shares significant similarities with exonucleases

COX et al. 281
FIGURE 2. Relatedness of CG6744 to 3′-5′ exonucleases. (A) Phylogenetic tree of
CG6744 relatedness (BLASTP search was carried out using the CG6744 predicted protein
sequence; “ unnamed protein product” highlighted is the CG6744 sequence used in the
search). (B) Radial tree, showing only a subset of CG6744-related proteins for clarity.
CG6744 and human EXDL2 (uncharacterized predicted 3′-5′ exonuclease domain-like 2
protein) are boxed.
both from lower eukaryotes (e.g., Tetrahymena, Dictyostelium) and higher
organisms (e.g., human EXDL2).
The lack of a Drosophila RecQ helicase with associated exonuclease domain
leads us to suggest that flies use a RecQ-family helicase in combination with
an exonuclease, as in E. coli and other lower eukaryotes (e.g., Arabidopsis).

282 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES
FIGURE 3. Insertional mutation of CG6744. (A) The position of the piggyBac inser-
tional c05871 element within the CG6744 gene is shown. Note the insert is 7.25 kb while
the CG6744 mRNA is 1947 nt. (B) Reverse transcription PCR from total RNA. Lanes 1, 2:
CG6744c05871 homozygous flies; lanes 3, 4: heterozygous flies; lanes 5, 6: wild-type flies.
Lanes 1, 3, 5 are males and lanes 2, 4, 6 are females. M= size marker and N= negative con-
trol lacking RNA template. Upper panel: CG6744, using primers designed across splice
sites such that they will amplify only correctly spliced product. Lower panel: actin 5C
control, using primers specific to Drosophila actin 5C.
Which helicase and nuclease is used in this context is not yet known, and it is
possible that several combinations can occur within cells.
Insertional Mutation of CG6744 Decreases Its Expression
To investigate the phenotypic effects of defects in CG6744, we obtained
flies carrying a mutation in CG6744. The mutant allele, CG6744c05871, results
from the insertion of a 7.25 kb piggyBac element48 within intron 2 (FIG. 3A).
Levels of CG6744 mRNA were assessed by reverse transcription PCR from
homozygous mutant flies compared with heterozygotes and wild-type flies.
From FIGURE 3B, it is apparent that mRNA levels of CG6744 are decreased in
flies homozygous for the piggyBac insertion (note the absence of product in the
negative control lacking template RNA). Actin controls (lower panel FIG. 3B)
demonstrate that this decrease is specific to CG6744 and not a result of global
differences in mRNA levels between the fly populations. The primers were
designed to amplify only correctly spliced mature mRNA; it is conceivable

COX et al. 283
that the large insertion in intron 2 disrupts either transcription (since the insert
also bears its own promoter and transcriptional termination sequences48) or
splicing, although any splicing defect cannot be fully penetrant since some
mRNA product is observed in the mutant flies. It is important to note that flies
homozygous for this CG6744 mutation show no apparent anatomical defects
at the gross level.
Recombination Is Elevated in CG6744 Mutants
Loss of WRN function leads to hyper-recombination in human cells11–13 and
mutation of the WRN exonuclease-like CG7670 leads to very high rates of
somatic recombination in Drosophila.47 Given the similarities between WRN,
CG7670, and CG6744, we tested the possibility that mutation of CG6744 gives
rise to excessive recombination.
Mitotic recombination can be easily scored in Drosophila by using suitable
marker mutations. One such marker is the mutant multiple wing hairs (mwh).
Animals homozygous for the recessive allele mwh1 develop a tuft of hairs,
rather than the normal single hair, on each cell of the wing blade. The multiple
wing-hair phenotype is cell autonomous, and mwh1/mwh1 mutant animals are
fully viable. Recombination or chromosome breakage between mwh1 (which
lies near the tip of chromosome arm 3L) and the centromere leads to clones
of cells homozygous for mwh1, which display the multiple wing-hair pheno-
type in a background of normal cells. To analyze levels of recombination or
chromosome breakage in the CG6744 mutant flies, we generated w1118; mwh1
CG6744c05871/CG6744c05871 flies. These flies demonstrated clusters of cells
with abnormal wing hairs (FIG. 4A). We have counted the number of cells in
such clusters and from the number of cells, calculated the number of cell divi-
sions that have taken place since the cells became homozygous or hemizygous
for mwh1 (FIG. 4B). The overall clone incidence (2.8 per fly) is markedly higher
than that in flies wild type for CG6744 (0.2 per fly), but considerably lower
than the frequency seen in similar experiments analyzing the CG7670 exonu-
clease (100–150 clones per fly47). This difference in recombination frequency
from that observed in CG7670 mutant flies47 may simply reflect differences in
expression (the CG7670 mutant is severely hypomorphic47 while expression is
decreased but not lost in the CG6744 piggyBac mutants used here). However,
it is also possible that the genes are differentially expressed in distinct cell
lineages, perhaps somatic versus germline cells. This is consistent with tissue-
specific differences in Drosophila RecQ helicase expression (e.g., RECQE is
expressed in early embryos49).
Two main mechanisms are possible for the generation of the mwh clones ob-
served on the wing blades of CG6744 mutant flies: homologous recombination
and chromosome breakage. We cannot, at this stage, distinguish between either
of these possibilities or nonhomologous end joining (NHEJ). A role for WRN

284 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES
FIGURE 4. Recombination in CG6744c05871 mutant flies. The mwh marker was used to
determine frequency of recombination in the developing wing blade. (A) Photomicrographs
showing typical mwh1 clones from CG6744 mutant flies; the large panel shows a clone of
many cells arising by a recombination event that occurred approximately five cell divisions
prior to wing-blade differentiation, while the smaller panel on the right shows a single cell
with the mwh phenotype, suggesting that recombination occurred in this cell just prior to
cessation of cell division. (B) Frequency of wing-blade clones on CG6744 mutant flies is
plotted as a function of number of cell cycles since that clone arose (n= 11; mean = 2.82
clones per fly).
in NHEJ has been deduced from the finding of large deletions in a plasmid-
based assay in transfected WS cells, association between WRN and Ku,50,51
and very recently, suppression of CPT hypersensitivity in WS cells by deletion
of Ku or DNA-PKcs.52 It will, therefore, be interesting in the future to assess
CPT sensitivity of CG6744 mutant flies and to analyze possible deletions by
sequence analysis at artificially induced DNA breaks.
Overall, we have shown that Drosophila melanogaster possesses two can-
didate WRN-like exonucleases, together with three RecQ-like helicases.
We suggest that reconstitution of WRN activity in flies occurs through
association of a RecQ helicase with a WRN-like exonuclease. We find hyper-
recombination on mutation of either of the putative WRN exonucleases,
CG767047 or CG6744 (this work), consistent with the hyper-recombinant

COX et al. 285
phenotype of human cells lacking WRN and the importance of WRN ex-
onuclease in DNA end processing in recombination, repair, and possibly DNA
replication.8,37
MATERIALS AND METHODS
Bioinformatics Searches
BLAST searches43 were conducted against Release 4.0 of the Drosophila
melanogaster genome sequence. Pairwise alignments were conducted with
BLASTP using human WRN as query against the Drosophila Refseq protein
database, and either CG6744 or CG7670 against the human Refseq protein
database (default parameters). T-coffee was performed at the European Bioin-
formatics Institute (Cambridge, UK; available from http://www.ebi.ac.uk/t-
coffee/). Phylogenetic trees were derived from BLASTP search results queried
with CG6744; a subset of homologues were selected manually for the radial
plot representing a range of phyla.
Fly Manipulation
Fly stocks were obtained from the Bloomington Drosophila Stock Center
(Bloomington, IN; http://flystocks.bio.indiana.edu/) and were maintained on
a standard oatmeal, yeast, molasses, and agar medium. Wing blades were dis-
sected from flies stored in 70% ethanol, mounted in Gary’s Magic Mountant,53
and analyzed by brightfield microscopy.
Reverse Transcription PCR
Gene-specific primers designed to CG6744 or actin 5C were used
in One-step RT-PCR (Qiagen Ltd., Crawley, West Sussex, UK; http:
//www1.qiagen.com/) on 0.5 g total RNA extracted from flies using Qiagen
RNeasy according to manufacturer’s instructions. Primers used were: CG6744-
Ex1/2-F 5′ TGAATGAACTGAAGAATCACTG 3′, CG6744-1333R 5′ TCGC-
CATGCTGACAGTAGTC 3′; actin 5C F 5′CACCGGTATCGTTCTGGACT3′,
actin 5C R 5′GGACTCGTCGTACTCCTGCT3′. Products were analyzed on
0.9% agarose gels stained with ethidium bromide against a 100 bp ladder
(Roche, Lewes, East Sussex, UK; http://www.roche.com).
ACKNOWLEDGMENTS
We thank Mrs. Christine Borer for technical support and the BBSRC
(Biotechnology and Biological Sciences Research Council, UK) for funding

286 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES
this work: I.B. and D.J.C. are supported by grants BB/E000924/1 to L.S.C. and
BB/E002072/1 to R.D.C.S., respectively.
REFERENCES
1. EPSTEIN, C.J. et al. 1966. Werner’s syndrome a review of its symptomatology,
natural history, pathologic features, genetics and relationship to the natural aging
process. Medicine (Baltimore) 45: 177–221.
2. GOTO, M. & R.W. MILLER. 2001. From Premature Gray Hair to Helicase-Werner
Syndrome: implications for Aging and Cancer. Japan Scientific Societies Press.
Tokyo.
3. COX, L.S. & R.G.A. FARAGHER. 2007. From old organisms to new molecules:
integrative biology and therapeutic targets in accelerated human ageing. Cell
and Molecular Life Sciences. E-pub ahead of print Jul 30, DOI 10.1007/S00018-
007-7123-x.
4. YU, C.E. et al. 1996. Positional cloning of the Werner’s syndrome gene. Science
272: 258–262.
5. GRAY, M.D. et al. 1997. The Werner syndrome protein is a DNA helicase. Nat.
Genet. 17: 100–103.
6. HUANG, S. et al. 1998. The premature ageing syndrome protein, WRN, is a 3′-5′
exonuclease. Nat. Genet. 20: 114–116.
7. XUE, Y. et al. 2002. A minimal exonuclease domain of WRN forms a hexamer on
DNA and Possesses both 3′-5′ exonuclease and 5′-protruding strand endonucle-
ase activities. Biochemistry 41: 2901–2912.
8. PERRY, J.J. et al. 2006. WRN exonuclease structure and molecular mechanism
imply an editing role in DNA end processing. Nat. Struct. Mol. Biol. 13: 414–
422.
9. BACHRATI, C.Z. & I.D. HICKSON. 2003. RecQ helicases: suppressors of tumorige-
nesis and premature aging. Biochem. J. 374: 577–606.
10. FARAGHER, R.G. et al. 1993. The gene responsible for Werner syndrome may be a
cell division “counting” gene. Proc. Natl. Acad. Sci. USA 90: 12030–12034.
11. SALK, D. et al. 1981. Cytogenetics of Werner’s syndrome cultured skin fibroblasts:
variegated translocation mosaicism. Cytogenet. Cell Genet. 30: 92–107.
12. SCAPPATICCI, S., D. CERIMELE & M. FRACCARO. 1982. Clonal structural chromoso-
mal rearrangements in primary fibroblast cultures and in lymphocytes of patients
with Werner’s Syndrome. Hum. Genet. 62: 16–24.
13. FUKUCHI, K., G.M. MARTIN & R.J. MONNAT, JR. 1989. Mutator phenotype of
Werner syndrome is characterized by extensive deletions. Proc. Natl. Acad. Sci.
USA 86: 5893–5897.
14. POOT, M. et al. 1992. Impaired S-phase transit of Werner syndrome cells expressed
in lymphoblastoid cell lines. Exp. Cell Res. 202: 267–273.
15. PICHIERRI, P. et al. 2001. Werner’s syndrome protein is required for correct recovery
after replication arrest and DNA damage induced in S-phase of cell cycle. Mol.
Biol. Cell 12: 2412–2421.
16. RODRIGUEZ-LOPEZ, A.M. et al. 2002. Asymmetry of DNA replication fork pro-
gression in Werner’s syndrome. Aging Cell 1: 30–39.
17. PICHIERRI, P. et al. 2000. Werner’s syndrome cell lines are hypersensitive to
camptothecin-induced chromosomal damage. Mutat. Res. 456: 45–57.

COX et al. 287
18. POOT, M., K.A. GOLLAHON & P.S. RABINOVITCH. 1999. Werner syndrome lym-
phoblastoid cells are sensitive to camptothecin-induced apoptosis in S-phase.
Hum. Genet. 104: 10–14.
19. LOWE, J. et al. 2004. Camptothecin sensitivity in Werner syndrome fibroblasts as
assessed by the COMET technique. Ann. N. Y. Acad. Sci. 1019: 256–259.
20. PRINCE, P.R. et al. 1999. Cell fusion corrects the 4-nitroquinoline 1-oxide sensitivity
of Werner syndrome fibroblast cell lines. Hum. Genet. 105: 132–138.
21. RODRIGUEZ-LOPEZ, A.M. et al. 2007. Correction of proliferation and drug sensitiv-
ity defects in the progeroid Werner’s syndrome by Holliday junction resolution.
Rejuvenation Research 10: 27–40.
22. HICKSON, I.D. 2003. RecQ helicases: caretakers of the genome. Nat. Rev. Cancer
3: 169–178.
23. SAINTIGNY, Y. et al. 2002. Homologous recombination resolution defect in werner
syndrome. Mol. Cell Biol. 22: 6971–6978.
24. OPRESKO, P.L. et al. 2003. Werner syndrome and the function of the Werner protein;
what they can teach us about the molecular aging process. Carcinogenesis 24:
791–802.
25. CHENG, W.H. et al. 2004. Linkage between Werner syndrome protein and the
Mre11 complex via Nbs1. J. Biol. Chem. 279: 21169–21176.
26. MASSIP, L. et al. 2006. Increased insulin, triglycerides, reactive oxygen species,
and cardiac fibrosis in mice with a mutation in the helicase domain of the Werner
syndrome gene homologue. Exp. Gerontol. 41: 157–168.
27. CHANG, S. et al. 2004. Essential role of limiting telomeres in the pathogenesis of
Werner syndrome. Nat. Genet. 36: 877–882.
28. COURCELLE, J. & P.C. HANAWALT. 1999. RecQ and RecJ process blocked replication
forks prior to the resumption of replication in UV-irradiated Escherichia coli.
Mol. Gen. Genet. 262: 543–551.
29. HARTUNG, F., H. PLCHOVA & H. PUCHTA. 2000. Molecular characterisation of RecQ
homologues in Arabidopsis thaliana. Nucleic Acids Res. 28: 4275–4282.
30. HARTUNG, F. & H. PUCHTA. 2006. The RecQ gene family in plants. J. Plant Physiol.
163: 287–296.
31. PLCHOVA, H., F. HARTUNG & H. PUCHTA. 2003. Biochemical characterization of
an exonuclease from Arabidopsis thaliana reveals similarities to the DNA ex-
onuclease of the human Werner syndrome protein. J. Biol. Chem. 278: 44128–
44138.
32. KETTING, R.F. et al. 1999. Mut-7 of C. elegans, required for transposon silencing
and RNA interference, is a homolog of Werner syndrome helicase and RNaseD.
Cell 99: 133–141.
33. LEE, S.J. et al. 2004. A Werner syndrome protein homolog affects C. elegans
development, growth rate, life span and sensitivity to DNA damage by acting at
a DNA damage checkpoint. Development 131: 2565–2575.
34. KATO, A. & H. INOUE. 2006. Growth defect and mutator phenotypes of RecQ-
deficient Neurospora crassa mutants separately result from homologous recom-
bination and nonhomologous end joining during repair of DNA double-strand
breaks. Genetics 172: 113–125.
35. HOPE, J.C. et al. 2006. Rqh1 blocks recombination between sister chromatids
during double strand break repair, independent of its helicase activity. Proc.
Natl. Acad. Sci. USA 103: 5875–5880.
36. AHMAD, F., C.D. KAPLAN & E. STEWART. 2002. Helicase activity is only partially
required for Schizosaccharomyces pombe Rqh1p function. Yeast 19: 1381–1398.

288 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES
37. KASHINO, G. et al. 2005. Exogenous expression of exonuclease domain-deleted
WRN interferes with the repair of radiation-induced DNA damages. J. Radiat.
Res. (Tokyo) 46: 407–414.
38. WU, L. et al. 2005. The HRDC domain of BLM is required for the dissolution of
double Holliday junctions. Embo J. 24: 2679–2687.
39. ZHANG, R. et al. 2005. BLM helicase facilitates Mus81 endonuclease activity in
human cells. Cancer Res. 65: 2526–2531.
40. SWANSON, C. et al. 2004. The Werner syndrome protein has separable recombina-
tion and survival functions. DNA Repair (Amst.) 3: 475–482.
41. CRABBE, L. et al. 2004. Defective telomere lagging strand synthesis in cells lacking
WRN helicase activity. Science 306: 1951–1953.
42. ADAMS, M.D. et al. 2000. The genome sequence of Drosophila melanogaster.
Science 287: 2185–2195.
43. ALTSCHUL, S.F. et al. 1997. Gapped BLAST and PSI-BLAST: a new generation
of protein database search programs. Nucleic Acids Res. 25: 3389–3402.
44. KUSANO, K., M.E. BERRES & W.R. ENGELS. 1999. Evolution of the RECQ family
of helicases: a drosophila homolog, Dmblm, is similar to the human bloom
syndrome gene. Genetics 151: 1027–1039.
45. SEKELSKY, J.J. et al. 1999. Drosophila and human RecQ5 exist in different isoforms
generated by alternative splicing. Nucleic Acids Res. 27: 3762–3769.
46. SEKELSKY, J.J., M.H. BRODSKY & K.C. BURTIS. 2000. DNA repair in Drosophila:
insights from the Drosophila genome project. J. Cell Biol. 150: F31–F36.
47. SAUNDERS, R.D. et al. 2007. Identification and characterisation of a Drosophila
ortholog of WRN exonuclease that is required to maintain genome integrity.
Aging Cell In press.
48. THIBAULT, S.T. et al. 2004. A complementary transposon tool kit for Drosophila
melanogaster using P and piggyBac. Nat. Genet. 36: 283–287.
49. JEONG, S.M. et al. 2000. Identification of Drosophila melanogaster RECQE as a
member of a new family of RecQ homologues that is preferentially expressed in
early embryos. Mol. Gen. Genet. 263: 183–193.
50. LI, B. et al. 2004. Identification and biochemical characterization of a Werner’s
syndrome protein complex with Ku70/80 and poly(ADP-ribose) polymerase-1.
J. Biol. Chem. 279: 13659–13667.
51. LI, B. et al. 2005. A conserved and species-specific functional interaction be-
tween the Werner syndrome-like exonuclease atWEX and the Ku heterodimer in
Arabidopsis. Nucleic Acids Res. 33: 6861–6867.
52. OTSUKI, M. et al. 2007. WRN counteracts the NHEJ pathway upon camptothecin
exposure. Biochem. Biophys. Res. Commun. 355: 477–482.
53. LAWRENCE, P., P. JOHNSTON & G. MORATA. 1986. Methods of marking cells. In
Drosophila: A Practical Approach. D.B. Roberts, Ed.: 229–242. IRL Press. Ox-
ford.
54. KUSANO, K., D.M. JOHNSON-SCHLITZ & W. ENGELS. 2001. Sterility of Drosophila
with mutations in the Bloom syndrome gene- complementation by Ku70. Science
291: 2600–2602.