Download DNA helicase deficiencies associated with cancer

© 2001 Oxford University Press
Human Molecular Genetics, 2001, Vol. 10, No. 7
741–746
DNA helicase deficiencies associated with cancer
predisposition and premature ageing disorders
Payam Mohaghegh and Ian D. Hickson+
Imperial Cancer Research Fund Laboratories, University of Oxford, Weatherall Institute of Molecular Medicine,
John Radcliffe Hospital, Oxford OX3 9DS, UK
Received 10 January 2001; Accepted 25 January 2001
Deficiency in a helicase of the RecQ family is found in at least three human genetic disorders associated with
cancer predisposition and/or premature ageing. The RecQ helicases encoded by the BLM, WRN and RECQ4
genes are defective in Bloom’s, Werner’s and Rothmund–Thomson syndromes, respectively. Cells derived
from individuals with these disorders in each case show inherent genomic instability. Recent studies have
demonstrated direct interactions between these RecQ helicases and human nuclear proteins required for several
aspects of chromosome maintenance, including p53, BRCA1, topoisomerase III, replication protein A and
DNA polymerase δ. Here, we review this network of protein interactions, and the clues that they present
regarding the potential roles of RecQ family members in DNA repair, replication and/or recombination pathways.
INTRODUCTION
DNA damage exists in many different forms and can arise as a
result of defects in any aspect of DNA metabolism. To deal with
DNA damage, all cells have developed a range of responses,
which include pathways for DNA repair and arrest of the cell
cycle (1,2). Mutations in DNA repair genes frequently lead to
genome destabilization and a consequent increase in the
frequency of mutations at other loci. Hence, germline mutations
in genes involved in DNA repair could result in the appearance
of a broad spectrum of disease phenotypes. In particular, the
general maintenance of genome stability is crucial for the prevention of cancer because of a requirement to maintain the integrity
of proto-oncogenes and tumour suppressor genes. This genome
maintenance is achieved by mechanisms that eliminate, with
high fidelity, DNA damage that occurs spontaneously, through
the action of DNA reactive agents, or through intrinsic errors in
DNA metabolism itself. One common feature of many processes
in DNA metabolism is a requirement for the complementary
strands of the DNA duplex to be separated through the action of
a DNA helicase enzyme. DNA helicases utilize the energy
derived from hydrolysis of ATP to perform essential roles in the
processes of genetic recombination, transcription, DNA replication and DNA repair (3,4). Indeed, as much as 1% of eukaryotic
genes may encode DNA and/or RNA helicase enzymes (5). In
this review, we discuss recent findings linking deficiency in one
family of DNA helicase enzymes, the RecQ family, to pathways
essential for the prevention of tumorigenesis and premature
ageing.
genetic recombination (6). Mutations of genes in this pathway
lead to deficiency in conjugational recombination and sensitivity
to UV light when the major pathway for recombination, defined
by the RecBCD helicase, is impaired. However, in an otherwise
wild-type background, recQ mutants show an elevated
frequency of illegitimate recombination (7). The RecQ family
includes representatives in prokaryotes and unicellular eukaryotes, as well as in vertebrates (Fig. 1) (8,9). RecQ family
members share a highly conserved domain comprising approximately 450 amino acids, which includes seven sequence motifs
found in many classes of DNA and RNA helicases. Amongst
these motifs is an ATP binding sequence (the so-called Walker
A-box) and a Dex H-box, which is a characteristic of the RecQ
family. Outside this domain, there is only limited sequence similarity amongst the family members. Where studied, RecQ family
members have been shown to be DNA helicases that translocate
in the 3′→5′ direction (10–17). WRN is unique in also being a
3′→5′ exonuclease dependent upon a functionally separable
domain in the N-terminal region of the protein (Fig. 1) (18–20).
Recent data indicate that mutations in genes encoding helicases
are responsible for genomic instability disorders of man. The
functional importance of RecQ family helicases in the maintenance of genome integrity is highlighted by the identification
of five human RecQ family helicases, three of which are
involved in autosomal recessive genomic instability disorders
associated with cancer predisposition and/or premature ageing.
These disorders, which are discussed in detail below are
Bloom’s syndrome (BS), Werner’s syndrome (WS) and Rothmund–Thomson syndrome (RTS).
RecQ FAMILY HELICASES
HUMAN RecQ HELICASE-DEFICIENT DISORDERS
The RecQ family is named after the RecQ protein of Escherichia
coli, which is a component of the so-called RecF pathway of
The BLM gene (21) is mutated in BS, a rare disorder associated
with pleiotropic phenotypes including immunodeficiency,
+To
whom correspondence should be addressed. Tel: +44 1865 222417; Fax: +44 1865 222431; Email: [email protected]
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Human Molecular Genetics, 2001, Vol. 10, No. 7
Figure 1. Schematic representation of the RecQ family of DNA helicases from bacteria (E.coli), yeast (Saccharomyces cerevisiae, Schizosaccharomyces pombe),
fruit fly (Drosophila melanogaster), frog (Xenopus laevis) and human (Homo sapiens). The central conserved helicase domain is shown as a blue box. The exonuclease domain of WRN is shown as a yellow box. The grey boxes denote domains with only a limited sequence homology. The nuclear localization signal
sequence (NLS) in BLM and WRN are shown in black and marked above. The RECQ5β protein is the largest of three possible variants generated by differential
RNA splicing. The size of each protein (in amino acids) is shown on the right.
Table 1. Human RecQ helicase-deficient disorders and RecQ helicase-interacting proteins
Syndrome
Phenotype
Interacting proteins
Features of normal ageing
Premature greying and thinning of the hair, loss of skin
elasticity, cataracts, type II diabetes mellitus, hypogonadism,
osteoporosis and atherosclerosis
Human RPA, p53, DNA polymerase δ,
PCNA, topoisomerase I, Ku70 and Ku86,
Ubc9 (SUMO-1 covalent modification)
Features not seen in normal
ageing
Unusual pattern of loss of subcutaneous fat, skin ulceration.
Cancer-prone, in particular an elevated incidence of
sarcomas
Werner’s
Bloom’s
Immuno-deficiency, male infertility and female sub-fertility,
small body size (proportional), sun-induced facial erythema,
and a predisposition to cancers of all types.
Human RPA, topoisomerase IIIα and
RAD51.The BASC complex: (which
includes ATM, RAD50, MSH6, RCF140,
BRCA1)
Rothmund–Thomson
Growth deficiency, photosensitivity with poikilodermatous
skin changes, early greying and hair loss, cataracts, increase
in cancer incidence, mainly osteogenic sarcomas
None identified
impaired fertility, proportional dwarfism, sun-induced facial
erythema and a predisposition to cancer (mean age at cancer
diagnosis of approximately 24 years) (22,23). This disorder is
of particular interest because affected individuals are susceptible to the full range of cancers seen in the normal population.
BS cells show a high frequency of genetic recombination
events, particularly sister chromatid exchanges (SCEs) and an
elevated rate of somatic mutation (9,22) (Table 1).
Mutations in the WRN gene (24) give rise to WS, which is
associated at a relatively early age with many, but not all, of the
features of the normal ageing process (25). Hence, WS individuals show many age-related disorders that develop from
puberty, including greying and thinning of the hair, cataracts,
type II diabetes mellitus, osteoporosis and atherosclerosis.
Moreover, WS individuals are also cancer-prone, although to a
more limited extent than is seen in BS individuals, in particular
displaying an elevated incidence of sarcomas (Table 1).
Recently, it has been shown that the RECQ4 gene (26) is
mutated in some cases of RTS. In this disorder, affected
individuals show growth deficiency, photosensitivity with
Human Molecular Genetics, 2001, Vol. 10, No. 7
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Table 2. Roles of WRN in cellular responses to DNA damage, and evidence of its presence at sites of DNA replication
WRN translocates to sites of replication/repair when replication is blocked by hydroxyureaa
The X.laevis WRN homologue, foci-forming activity-1 (FFA-1), has been shown to be essential for the formation of replication foci
containing replication ‘factories’b
WRN copurifies with DNA replication complexes and interacts with PCNA and topoisomerase Ic
The heterotrimeric, single-stranded DNA binding protein, RPA, interacts with WRN, and its presence increases WRN helicase activityd
WS cells show a prolonged S phasee
WS cells show hypersensitivity to 4NQO which forms covalent adducts, but also possibly oxidative DNA damage including strand
breaksf
WRN helicase activity is inhibited by DNA minor groove binding drugs distamycin A and netrospig
WRN interacts functionally with DNA polymerase δ. This polymerase is required for DNA replication and repair in eukaryotesh
aConstantinou
et al. (33).
et al. (34).
cLebel et al. (35).
dBrosh et al. (36).
ePoot et al. (37).
fOgburn et al. (38).
gBrosh et al. (39).
hKamath-Loeb et al. (40).
bYan
poikilodermatous skin changes, cataracts, early greying and
loss of hair, as well as some increase in cancer incidence (27).
As with WS, the cancer predispositon in RTS individuals is of
a limited range, mainly osteogenic sarcomas (Table 1).
One feature that links these three genetic disorders at the
cellular level is inherent genomic instability (9). In the case of
BS cells, this instability is manifested as a 10-fold elevated
frequency of homologous recombination events, including
reciprocal exchanges between sister-chromatids and homologous chromosomes (9,22,23). WS cells do not show elevated
SCE frequencies, but they do display increased illegitimate
recombination and a high frequency of large chromosomal
deletions (25). The genomic instability of RTS cells has not
been analysed in detail, but there are reports of an increased
frequency of chromosome aberrations (28).
PROTEINS INTERACTING WITH HUMAN RecQ
FAMILY HELICASES
The last year or so has seen the identification of numerous
proteins that interact physically with human RecQ family
members. Although in some cases the functional significance
of these interactions remains to be identified, there is already
sufficient evidence to indicate that RecQ helicases are important components of DNA repair, replication and recombination
pathways (Table 1). Below, we review evidence that RecQ
family helicases form complexes with other proteins involved
in these processes.
WRN-INTERACTING PROTEINS
WRN has been shown to form complexes with proteins
involved both in cellular responses to DNA damage and in
DNA replication (Table 2). The identification of a functional
interaction between WRN and the p53 tumour suppressor
protein serves to emphasize the role of the RecQ family in the
maintenance of genomic stability (29,30). Mutations in p53,
which has been dubbed the ‘guardian of the genome’, are seen
in >50% of all sporadic cancers in humans. p53 functions in a
highly dynamic and controlled manner; induction of p53 leads
to cell cycle arrest in G1 and/or G 2, allowing time for DNA
repair to take place, but may additionally lead to apoptotic cell
death (1,2). Moreover, the loss of p53 results in genomic instability. In WS cells, p53-mediated apoptosis is attenuated, while
ectopic expression of WRN in these cells can rescue this deficiency (29). Overexpression of WRN results in elevated p53dependent transcriptional activity and induction of p21Waf1
(29,30). The interaction between p53 and WRN takes place via
their respective C-terminal domains. Interestingly, this is the
region where the majority of missense mutations found in WS
patients are located (25).
Without detailed knowledge of the cellular functions of
WRN, any discussion about the potential roles for the p53–
WRN complex is necessarily speculative. However, there is
increasing evidence to suggest that WRN acts at DNA replication origins or at sites of blocked replication forks (see discussion below and Table 2). Hence loss of the p53/WRN
interaction could result in WRN being unable to recruit p53 to
replication origins/forks in response to DNA damage. In
normal cells, the p53–WRN complex may recognize abnormal
DNA structures, such as stalled replication forks, leading
either to a coordination of cell cycle and DNA repair events or
to the induction of apoptosis. There are two possible routes for
repair at this stage with a requirement for WRN. First, WRN
could be involved directly in restoration of DNA replication by
displacing (perhaps abnormal?) Okazaki fragments on the
lagging strand and degrading the displaced DNA. Recently, it
has been shown that the WRN exonuclease catalyses structuredependent degradation of DNA, suggesting that WRN resolves
abnormal DNA structures via both its helicase and its exonuclease activities (31). Consistent with this notion, a recent
paper by Cooper et al. (32) supports a role for the WRN exonu-
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clease activity during repair processes. It was shown the Ku70
and Ku86 DNA end-binding complex directly interacts with
WRN, and stimulates its 3′→5′ exonuclease activity.
A second possible role for WRN in replication fork repair
would be after removal of the damaged DNA strand at blocked
forks. For example, it is possible that RAD51 (the human
RecA homologue) could stabilize the replication fork at this
stage, allowing the continuation of DNA synthesis without a
need for re-initiation of replication (25). Alternatively, WRN
could be involved in repair at blocked forks via homologous
recombination; the role of WRN in this case could be to
promote translocation of Holliday junctions, and prevent aberrant recombination events at sites of stalled replication forks
by dissociating recombination intermediates (33). Indeed,
there is now considerable evidence supporting a role for WRN
in cellular response to DNA damage, and its presence at sites
of DNA replication (Table 2).
The interaction of WRN with DNA polymerase δ (Table 2)
provides a direct biochemical link between WRN and DNA
synthesis. It was shown that WRN increases the rate of nucleotide incorporation by DNA polymerase δ in the absence of
proliferating cell nuclear antigen (PCNA); however, WRN has
no significant stimulatory effect on the DNA polymerase δ
holoenzyme (polymerase δ–PCNA complex) (40). Therefore,
Kamath-Loeb et al. (40) suggested that WRN is unlikely to
function in normal processive DNA synthesis, which requires
the polymerase δ–PCNA complex. Rather, they speculated that
WRN may function in a replication restart pathway at sites
where damaged DNA/unusual DNA secondary structures have
blocked DNA replication and where the DNA replication
machinery has detached from the DNA. It has also been
shown that overexpressed WRN is able to recruit DNA
polymerase δ to the nucleolus, suggesting that WRN could be
involved in regulating the initiation and progression of DNA
replication by recruiting polymerase δ to particular sites of
DNA synthesis (41).
One potential way of regulating and coordinating the many
roles of WRN could be by small ubiquitin-related modifier
(SUMO) modification. Addition of SUMO to target proteins
can change their localization or their interaction with other
proteins. WRN has been shown to be covalently attached with
SUMO-1 via the conjugating enzyme Ubc9 (42). Ubc9 has
been shown to play a role in the degradation of certain proteins,
including S and M phase cyclins (43). As discussed in more
detail below in the section on BLM-interacting proteins,
SUMO-1 modification is linked in some cases to regulation of
intranuclear localization. SUMO modification is also involved
in regulating p53 function, through interactions involving the
Mdm2 protein and changes in the half-life of p53 (44). Potentially, SUMO modification of p53 and WRN could play a critical role in orchestrating the cross-talk between these proteins
and hence regulate pathways for the maintenance of genome
integrity.
BLM-INTERACTING PROTEINS
BRCA1 is the protein defective in some cases of hereditary
breast cancer susceptibility. The recent identification of BLM
as part of the BRCA1-associated genome surveillance complex
(BASC), links BLM with a number of tumour suppressor and
DNA damage repair proteins (45). The BASC complex
includes MSH2, MHS6, MLH1, ATM, BLM, the RAD50–
MRE11–NBS1 complex and DNA replication factor C. Many
components of this complex have roles in recognition of DNA
damage/unusual DNA structures, suggestive of this complex
performing some kind of ‘sensor’ role. To examine the role of
BLM within BASC, the subcellular localization of BLM and
BRCA1 was analysed before and after exposure to DNA
damaging agents. In untreated cells, BLM and BRCA1
colocalization was limited to a few bright nuclear foci.
However, after treatment with hydroxyurea or ionizing radiation, colocalization was greatly enhanced in those cells that
were in mid-to-late S phase or in G2. This could be indicative
of specific requirement for BLM/BRCA1 in replication/repair
of late replicating DNA. Consistent with a role for BLM
(possibly within BASC) in recognizing abnormal DNA structures, we have shown recently that BLM is able to unwind a
variety of unusual DNA structures, including G-quadruplex,
synthetic X-junctions (models for the Holliday junction),
bubbles and forked DNA (P. Mohaghegh et al., submitted for
publication).
Recent data indicate that BLM binds to the 70 kDa subunit of
the heterotrimeric, single-stranded DNA binding protein, replication protein A (RPA) (46). This interaction stimulates the
helicase activity of BLM (46). RPA is involved in DNA replication, repair and recombination, and can be detected on
meiotic prophase chromosomes where it appears to play a role
in both homologous synapsis and recombination. BLM and
RPA also colocalise in meiotic prophase nuclei of mouse spermatocytes (47). Potentially, BLM and RPA could work
together to unwind various DNA structures to facilitate DNA
recombination during meiosis, perhaps through resolving
recombination intermediates.
A role for BLM in recombinational repair has been proposed
recently by our group, as a result of the finding of a direct interaction between BLM and the RAD51 recombinase enzyme
(48). RAD51 is the eukaryotic homologue of the E.coli RecA
protein, which is vital for homologous recombination and
recombinational repair of DNA double-strand breaks. BLM
and RAD51 were found to interact directly in vitro and to coimmunoprecipitate from nuclear extracts. As discussed above,
BLM localizes to nuclear foci in response to DNA damage and
colocalizes with BRCA1. We have shown that BLM also
colocalizes with a subset of RAD51 nuclear foci, which have
been suggested to be sites of ongoing DNA repair. As with the
BLM/BRCA1 colocalization, the number of colocalising
BLM/RAD51 foci increases after treatment of cells with
ionizing radiation. The precise functional role for the BLM/
RAD51 interaction is not currently known. One possibility has
been suggested by the recent work of van Brabant et al. (49).
These authors showed that BLM preferentially binds to and
melts DNA D-loops that can be formed by RAD51. D-loop
structures model the initial intermediate formed during homologous recombination, and van Brabant et al. (49) suggested
that BLM and RAD51 may have antagonistic roles in dealing
with DNA structures arising at collapsed replication forks. A
second possibility is that the BLM–RAD51 complex exists to
coordinate different steps of homologous recombination. In
this context, we have shown recently that BLM binds to the
Holliday junction recombination intermediate and promotes
ATP-dependent branch migration of these junctions (50).
Human Molecular Genetics, 2001, Vol. 10, No. 7
Some, but not all, of the nuclear foci that include BASC,
BLM and RAD51 colocalize with promyelocytic leukemia
(PML) nuclear bodies. It was shown in three independent
reports (51–53) that BLM localises to PML bodies in the
nucleoplasm of normal cells. One report suggested, however,
that BLM localizes to the nucleolus during S phase (53). In
contrast, we have shown that BLM can be found in replication
foci during S phase (54). It has been shown that BS cell lines
bud out micronuclei during S phase (53), and micronuclei are
proposed to be part of a p53-dependent process in response to
the stalling of DNA replication forks. Interestingly, in PML–/–
cell lines, BLM fails to accumulate in nuclear foci, and in these
cell lines the level of SCEs is higher than in PML+/+ controls.
These results suggest that PML is required for the localization
of BLM to nuclear foci, pointing to PML being a regulator of
BLM (52), and to a role for the PML body in regulating
genome stability. The ability of PML to regulate BLM and
other PML interacting proteins is potentially controlled by
SUMO-1 modification of PML and other proteins, including
possibly BLM (51).
The first direct biochemical evidence for a role for BLM in
DNA replication was provided by studies with the BLM homologue in Xenopus (xBLM). Antibodies were raised against
xBLM and used in a series of immunodepletion experiments.
DNA replication was strongly inhibited (5- to 10-fold) in
Xenopus egg extracts depleted of xBLM. This inhibition was
rescued by the addition of recombinant xBLM (55). These
results, showing a critical role for xBLM in DNA replication
are apparently in contradiction to previous observations
showing that yeast sgs1 and rqh1 mutants, as well as human
BS cells lacking BLM, are viable and can perform near normal
DNA replication (8,9).
During DNA replication, DNA helicases separate the
complementary DNA strands; this process of unwinding
produces torsional stress in the DNA, which is relieved by
topoisomerases. It would not be surprising, therefore, if DNA
helicases were to operate in close conjuction with topoisomerases. This has been shown for certain RecQ helicases. For
example, E.coli RecQ helicase can unwind a covalently-closed
double-stranded DNA substrate, and this activity stimulates
E.coli topoisomerase III to fully catenate (interlink) two or
more plasmid molecules (56). BLM also interacts with topoisomerase III, via both the N- and C-terminal domains of BLM
(57). To date, only an interaction with one of the two human
topoisomerase III isoforms, TOPO IIIα, has been shown (57).
The finding of two binding sites on BLM for TOPO IIIα
suggests that these proteins might interact in a 1:2 ratio. Since
topoisomerase III enzymes only make single-stranded DNA
nicks, the incorporation of two topoisomerase molecules could
allow double-strand breaks to take place. This could help to
disrupt recombination intermediates between inappropriately
paired DNA strands.
In summary, our knowledge of the biochemical properties of
the RecQ helicases, in terms of enzymatic activities, substrate
preferences and binding partners, has improved considerably
in the last 2 years or so. Nevertheless, while there are tantalizing links between various RecQ family members and DNA
replication and repair, there is still no clear view as to the
precise role(s) that these enzymes play in any of these processes. Given the burgeoning interest in the human RecQ
745
family members involved in the suppression of cancer and premature ageing, we expect the current level of ignorance to be
relatively short-lived.
ACKNOWLEDGEMENTS
We thank members of the ICRF Genome Integrity Group for
useful discussions, Mrs J. Pepper for preparation of the manuscript and Dr C.J. Norbury for critical reading of the manuscript. Work in the authors’ laboratory is supported by the
Imperial Cancer Research Fund.
REFERENCES
1. Norbury, C.J. and Hickson, I.D. (2001) Cellular responses to DNA damage. Annu. Rev. Pharmacol. Toxicol., 41, 367–401.
2. Zhou, B.B. and Elledge, S.J. (2000) The DNA damage response: putting
checkpoints in perspective. Nature, 408, 433–439.
3. Lohman, T.M. and Bjornson, K.P. (1996) Mechanisms of helicase-catalyzed DNA unwinding. Annu. Rev. Biochem., 65, 169–214.
4. Villani, G. and Tanguy Le Gac, N. (2000) Interactions of DNA helicases
with damaged DNA: possible biological consequences. J. Biol. Chem.,
275, 33185–33188.
5. Gorbalenya, A.E. and Koonin, E.V. (1993) Helicases: amino acid
sequence comparisons and structure-function relationships. Curr. Opin.
Struct. Biol., 3, 419–429.
6. Nakayama, H., Nakayama, K., Nakayama, R., Irino, N., Nakayama, Y.
and Hannawalt, P.C. (1984) Isolation and genetic characterization of a thiamineless death-resistant mutant of Escherichia coli K12: Identification
of a new mutation (recQ1) that blocks the RecF recombination pathway.
Mol. Gen. Genet., 195, 474–480.
7. Hanada, K., Ukita, T., Kohno, Y., Saito, K., Kato, J. and Ikeda, H. (1997)
RecQ DNA helicase is a suppressor of illegitimate recombination in
Escherichia coli. Proc. Natl Acad. Sci. USA, 94, 3860–3865.
8. Chakraverty, R.K. and Hickson, I.D. (1999) Defending genome integrity
during DNA replication: a proposed role for RecQ family helicases.
Bioessays, 21, 286–294.
9. Karow, J.K., Wu, L. and Hickson, I.D. (2000) RecQ family helicases:
roles in cancer and aging. Curr. Opin. Genet. Dev., 10, 32–38.
10. Seki, M., Miyazawa, H., Tada, S., Yanagisawa, J., Yamaoka, T., Hoshino,
S., Ozawa, K., Eki, T., Nogami, M., Okumura, K. et al. (1994) Molecular
cloning of cDNA encoding human DNA helicase Q1 which has homology
to Escherichia coli RecQ helicase and localization of the gene at chromosome 12p12. Nucleic Acids Res., 22, 4566–4573.
11. Puranam, K.L. and Blackshear, P.J. (1994) Cloning and characterization
of RECQL, a potential human homologue of the Escherichia coli DNA
helicase RecQ. J. Biol. Chem., 269, 29838–29845.
12. Karow, J.K., Chakraverty, R.K. and Hickson, I.D. (1997) The Bloom’s
syndrome gene product is a 3′→5′ DNA helicase. J. Biol. Chem., 272,
30611–30614.
13. Gray, M.D., Shen, J.C., Kamath-Loeb, A.S., Blank, A., Sopher, B.L.,
Martin, G.M., Oshima, J. and Loeb, L.A. (1997) The Werner syndrome
protein is a DNA helicase. Nature Genet., 17, 100–103.
14. Suzuki, N., Shimamoto, A., Imamura, O., Kuromitsu, J., Kitao, S., Goto,
M. and Furuichi, Y. (1997) DNA helicase activity in Werner’s syndrome
gene product synthesized in a baculovirus system. Nucleic Acids Res., 25,
2973–2978.
15. Shen, J.C., Gray, M.D., Oshima, J. and Loeb, L.A. (1998) Characterization of Werner syndrome protein DNA helicase activity: directionality,
substrate dependence and stimulation by replication protein A. Nucleic
Acids Res., 26, 2879–2885.
16. Umezu, K. and Nakayama, H. (1993) RecQ DNA helicase of Escherichia
coli: Characterisation of the helix-unwinding activity with emphasis on
the effect of single-stranded DNA-binding protein. J. Mol. Biol., 230,
1145–1150.
17. Bennett, R.J., Sharp, J.A. and Wang, J.C. (1998) Purification and characterization of the Sgs1 DNA helicase activity of Saccharomyces cerevisiae.
J. Biol. Chem., 273, 9644–9650.
18. Huang, S., Li, B., Gray, M.D., Oshima, J., Mian, I.S. and Campisi, J.
(1998) The premature ageing syndrome protein, WRN, is a 3′→5′ exonuclease. Nature Genet., 20, 114–116.
746
Human Molecular Genetics, 2001, Vol. 10, No. 7
19. Shen, J.C., Gray, M.D., Oshima, J., Kamath-Loeb, A.S., Fry, M. and Loeb,
L.A. (1998) Werner syndrome protein. I. DNA helicase and dna exonuclease reside on the same polypeptide. J. Biol. Chem., 273, 34139–34144.
20. Suzuki, N., Shiratori, M., Goto, M. and Furuichi, Y. (1999) Werner syndrome helicase contains a 5′→3′ exonuclease activity that digests DNA
and RNA strands in DNA–DNA and RNA–DNA duplexes dependent on
unwinding. Nucleic Acids Res., 27, 2361–2368.
21. Ellis, N.A., Groden, J., Ye, T.Z., Straughen, J., Lennon, D.J., Ciocci, S.,
Proytcheva, M. and German, J. (1995) The Bloom’s syndrome gene product is homologous to RecQ helicases. Cell, 83, 655–666.
22. German, J. (1993) Bloom syndrome: A mendelian prototype of somatic
mutational disease. Medicine, 72, 393–406.
23. German, J. (1995) Bloom’s syndrome. Dermatol. Clin., 13, 7–18.
24. Yu, C., Oshima, J., Fu, Y., Wijsman, E.M., Hisama, F., Alisch, R.,
Matthews, S., Najura, J., Miki, T., Ouais, S. et al. (1996) Positional cloning of the Werner’s syndrome gene. Science, 272, 258–262.
25. Shen, J.C. and Loeb, L.A. (2000) The Werner syndrome gene: the molecular basis of RecQ helicase-deficiency diseases. Trends Genet., 16, 213–220.
26. Kitao, S., Shimamoto, A., Goto, M., Miller, R.W., Smithson, W.A.,
Lindor, N.M. and Furuichi, Y. (1999) Mutations in RECQL4 cause a subset of cases of Rothmund-Thomson syndrome. Nature Genet., 22, 82–84.
27. Vennos, E.M. and James, W.D. (1995) Rothmund-Thomson syndrome.
Dermatol. Clin., 13, 143–150.
28. Vasseur, F., Delaporte, E., Zabot, M.T., Sturque, M.N., Barrut, D.,
Savary, J.B., Thomas, L. and Thomas, P. (1999) Excision repair defect in
Rothmund Thomson Syndrome. Acta. Derm. Venereol., 79, 150–152.
29. Spillare, E.A., Robles, A.I., Wang, X.W., Shen, J.C., Yu, C.E., Schellenberg, G.D. and Harris, C.C. (1999) p53-mediated apoptosis is attenuated
in Werner syndrome cells. Genes Dev., 13, 1355–1360.
30. Blander, G., Kipnis, J., Leal, J.F., Yu, C.E., Schellenberg, G.D. and Oren,
M. (1999) Physical and functional interaction between p53 and the
Werner’s syndrome protein. J. Biol. Chem., 274, 29463–29469.
31. Shen, J.C. and Loeb, L.A. (2000) Werner syndrome exonuclease catalyzes
structure-dependent degradation of DNA. Nucleic Acids Res., 28, 3260–3268.
32. Cooper, M.P., Machwe, A., Orren, D.K., Brosh, R.M., Ramsden, D. and
Bohr, V.A. (2000) Ku complex interacts with and stimulates the Werner
protein. Genes Dev., 14, 907–912.
33. Constantinou, A., Tarsounas, M., Karow, J.K., Brosh, R.M., Bohr, V.A.,
Hickson, I.D. and West, S.C. (2000) Werner’s syndrome protein (WRN)
migrates Holliday junctions and co-localizes with RPA upon replication
arrest. EMBO Reports, 1, 80–84.
34. Yan, H., Chen, C.Y., Kobayashi, R. and Newport, J. (1998) Replication
focus-forming activity 1 and the Werner syndrome gene product. Nature
Genet., 19, 375–378.
35. Lebel, M., Spillare, E.A., Harris, C.C. and Leder, P. (1999) The Werner
syndrome gene product co-purifies with the DNA replication complex
and interacts with PCNA and topoisomerase I. J. Biol. Chem., 274,
37795–37799.
36. Brosh, R.M., Jr, Orren, D.K., Nehlin, J.O., Ravn, P.H., Kenny, M.K.,
Machwe, A. and Bohr, V.A. (1999) Functional and physical interaction
between WRN helicase and human replication protein A. J. Biol. Chem.,
274, 18341–18350.
37. Poot, M., Hoehn, H., Runger, T.M. and Martin, G.M. (1992) Impaired Sphase transit of Werner syndrome cells expressed in lymphoblastoid cell
lines. Exp. Cell Res., 202, 267–273.
38. Ogburn, C.E., Oshima, J., Poot, M., Chen, R., Hunt, K.E., Gollahon, K.A.,
Rabinovitch, P.S. and Martin, G.M. (1997) An apoptosis-inducing genotoxin differentiates heterozygotic carriers for Werner helicase mutations
from wild-type and homozygous mutants. Hum. Genet., 101, 121–125.
39. Brosh, R.M., Jr, Karow, J.K., White, E.J., Shaw, N.D., Hickson, I.D. and
Bohr, V.A. (2000) Potent inhibition of Werner and Bloom helicases by
DNA minor groove binding drugs. Nucleic Acids Res., 28, 2420–2430.
40. Kamath-Loeb, A.S., Johansson, E., Burgers, P.M. and Loeb, L.A. (2000)
Functional interaction between the Werner Syndrome protein and DNA
polymerase delta. Proc. Natl Acad. Sci. USA, 97, 4603–4608.
41. Szekely, A.M., Chen, Y.H., Zhang, C., Oshima, J. and Weissman, S.M.
(2000) Werner protein recruits DNA polymerase delta to the nucleolus.
Proc. Natl Acad. Sci. USA, 97, 11365–11370.
42. Kawabe, Y., Seki, M., Seki, T., Wang, W.S., Imamura, O., Furuichi, Y.,
Saitoh, H. and Enomoto, T. (2000) Covalent modification of the Werner’s
syndrome gene product with the ubiquitin-related protein, SUMO-1.
J. Biol. Chem., 275, 20963–20966.
43. Seufert, W., Futcher, B. and Jentsch, S. (1995) Role of a ubiquitinconjugating enzyme in degradation of S- and M-phase cyclins. Nature,
373, 78–81.
44. Hochstrasser, M. (2000) Biochemistry. All in the ubiquitin family. Science,
289, 563–564.
45. Wang, Y., Cortez, D., Yazdi, P., Neff, N., Elledge, S.J. and Qin, J.
(2000) BASC, a super complex of BRCA1-associated proteins involved
in the recognition and repair of aberrant DNA structures. Genes Dev.,
14, 927–939.
46. Brosh, R.M., Jr, Li, J.L., Kenny, M.K., Karow, J.K., Cooper, M.P.,
Kureekattil, R.P., Hickson, I.D. and Bohr, V.A. (2000) Replication protein
A physically interacts with the Bloom’s syndrome protein and stimulates
its helicase activity. J. Biol. Chem., 275, 23500–23508.
47. Walpita, D., Plug, A.W., Neff, N.F., German, J. and Ashley, T. (1999)
Bloom’s syndrome protein, BLM, colocalizes with replication protein A
in meiotic prophase nuclei of mammalian spermatocytes. Proc. Natl Acad.
Sci. USA, 96, 5622–5627.
48. Wu, L., Davies, S.L., Levitt, N.C. and Hickson, I.D. (2001) Potential role
for the BLM helicase in recombinational repair via a conserved interaction
with RADS1. J. Biol. Chem.,in press.
49. van Brabant, A.J., Ye, T., Sanz, M., German, I.J., Ellis, N.A. and
Holloman, W.K. (2000) Binding and melting of D-loops by the Bloom
syndrome helicase. Biochemistry, 39, 14617–14625.
50. Karow, J.K., Constantinou, A., Li, J.L., West, S.C. and Hickson, I.D.
(2000) The Bloom’s syndrome gene product promotes branch migration
of holliday junctions. Proc. Natl Acad. Sci. USA, 97, 6504–9508.
51. Ishov, A.M., Sotnikov, A.G., Negorev, D., Vladimirova, O.V., Neff, N.,
Kamitani, T., Yeh, E.T., Strauss, J.F., III and Maul, G.G. (1999) PML is
critical for ND10 formation and recruits the PML-interacting protein daxx
to this nuclear structure when modified by SUMO-1. J. Cell Biol., 147,
221–234.
52. Zhong, S., Hu, P., Ye, T.Z., Stan, R., Ellis, N.A. and Pandolfi, P.P. (1999)
A role for PML and the nuclear body in genomic stability. Oncogene, 18,
7941–7947.
53. Yankiwski, V., Marciniak, R.A., Guarente, L. and Neff, N.F. (2000)
Nuclear structure in normal and Bloom syndrome cells. Proc. Natl Acad.
Sci. USA, 97, 5214–5219.
54. Wu, L., Davies, S.L and Hickson, I.D. (2001) Roles of RecQ family helicases in the maintenance of genome stability. Cold Spring Harb. Symp.
Quant. Biol., in press.
55. Liao, S., Graham, J. and Yan, H. (2000) The function of xenopus Bloom’s
syndrome protein homolog (xBLM) in DNA replication. Genes Dev., 14,
2570–2575.
56. Harmon, F.G., DiGate, R.J. and Kowalczykowski, S.C. (1999) RecQ helicase and topoisomerase III comprise a novel DNA strand passage function: a conserved mechanism for control of DNA recombination. Mol.
Cell, 3, 611–620.
57. Wu, L., Davies, S.L., North, P.S., Goulaouic, H., Riou, J.F., Turley, H.,
Gatter, K.C. and Hickson, I.D. (2000) The Bloom’s syndrome gene product interacts with topoisomerase III. J. Biol. Chem., 275, 9636–9644.