Carcinogenesis Advance Access originally published online on March 28, 2003
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Carcinogenesis, Vol. 24, No. 5, 791-802,
May 2003
© 2003 Oxford University Press
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Werner syndrome and the function of the Werner protein; what they can teach us about the molecular aging process.
Laboratory of Molecular Gerontology, National Institute on Aging, NIH, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA
1 To whom correspondence should be addressed Email: vbhor{at}nih.gov
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Abstract |
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Werner syndrome (WS) is a hallmark premature aging disease, in which the patients appear much older than their chronological age, and exhibit many of the clinical signs and symptoms of normal aging at an early stage in life. They develop many age-associated diseases early in life including atherosclerosis, osteoporosis, cataracts and display a high incidence of cancer. WS is also marked by increased genomic instability, manifested as chromosomal alterations. Characterization and study of the Werner protein (WRN) suggests that it participates in several important DNA metabolic pathways, and that its primary function may be in DNA repair processes. Thus, the WRN protein represents an important link between defective DNA repair and the processes related to aging and cancer.
Abbreviations: AA-PML, ALT-associated PML bodies; BER, base excision repair; BS, Bloom syndrome; CPT, camptothecin; dRP, 5'-deoxyribose 5-phosphate; D-loop, displacement loop; DSB, double strand break; HR, homologous recombination; IR, ionizing radiation; NHEJ, non-homologous end joining; 4-NQO, 4-nitroquinoline 1-oxide; NTS, nucleolar targeting sequence; PCNA, proliferating cell nuclear antigen; topo I, topoisomerase I; UV, ultraviolet; WRN, Werner protein; WS, Werner syndrome
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Introduction |
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Werner syndrome (WS) is an autosomal recessive disorder that belongs to a category of diseases called human premature aging disorders. These diseases are also called segmental progerias, designating that these conditions are associated with many, but not all of the clinical characteristics seen in the normal aging process. An increasing number of human disorders belong to this disease category, including Rothmund–Thomson syndrome (RTS), progeria, Cockayne syndrome, xeroderma pigmentosum and Hutchinson–Guilford progeria. There is considerable interest in researching the underlying molecular pathology of these diseases, as this will provide insight into the disease mechanisms of both the specific disorder involved, and also most probably aspects of the normal aging process in humans.
WS can be regarded as a model system for the study of normal aging, as well as of age-associated diseases. WS patients display a remarkable number of clinical signs and symptoms associated with aging early in life; however, there are also differences in clinical appearances between WS and normal aging. The premature aging symptoms appear in the second or third decade of life and include graying of the hair, cataracts, osteoporosis, atherosclerosis and diabetes mellitus (type II) (1,2). In addition, WS patients display a high incidence of malignant neoplasms, particularly sarcomas; however, the major cause of death is myocardial infarction at a median age of 47 years. Other non-age-related symptoms include short stature, soft tissue calcification, hypogonadism and reduced fertility. In support of WS as a model system of normal aging, we have recently conducted an extensive expression array analysis of WS and normal, young and old fibroblasts, and we find that WS very strongly resembles normal aging with regard to expression patterns (K.J.Kyng et al., submitted for publication).
Some of the cellular characteristics of WS are shown in Figure 1. Most of these will be discussed later in the review, but a hallmark feature of the disease is the genomic instability that is manifested as recombinational changes, chromosomal alterations and attenuated apoptosis. There are several cellular characteristics of defects in DNA repair and replication, as well as transcriptional and telomere maintenance problems that have been noted. The complex clinical and cellular phenotypes of WS have made determining the molecular pathology of WS most challenging.
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The cloning of the gene defective in WS has permitted the development of new experimental approaches for defining the molecular pathology of WS. Several of these approaches have focused on defining the cellular role of the 167 kDa Werner protein (WRN) encoded by the gene defective in WS (3). Experimental strategies have included determining the intracellular location of WRN, characterizing the biochemical properties of WRN and identifying proteins, which interact physically and functionally with WRN. Since the publication of the first WRN protein partners in 1999, several additional interacting partners have been reported in the literature (Table I). While these proteins all appear to function at some level in maintaining the integrity of the genome and in DNA damage response, they participate in both distinct and overlapping cellular pathways involved in DNA metabolism. Therefore, discerning the precise function of WRN with these various partners in vivo is an obvious challenge, and requires investigation into the physiological importance of the various WRN protein interactions. This review will focus on the cellular and biochemical evidence for the participation of the WRN protein in various cellular pathways, together with the identified WRN interacting protein partners.
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WRN protein biochemistry and catalytic activities |
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 TopAbstractIntroduction WRN protein biochemistry and... Intracellular localization of...Roles for WRN in...WRN roles in p53-mediated...Function of WRN in...Telomere metabolismPerspectiveReferences |
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The WRN protein is a member of the RecQ family of DNA helicases that includes proteins defective in the human genomic instability diseases Bloom syndrome (BS) (4), RTS (5) and others (Figure 2). WRN possesses DNA-dependent ATPase, 3'
5' helicase and 3'5' exonuclease activities (6–8). The substrate specificity of the WRN helicase in vitro includes a variety of DNA replication, recombination and repair intermediates including Holliday junctions and forked duplex structures (recently reviewed in refs 9,10) (Figure 3). RNA–DNA heteroduplex structures are also unwound by WRN (11). However, the WRN helicase unwinds substrates with limited processivity in vitro. WRN is unique among the human RecQ helicases identified thus far, in that it also contains an exonuclease domain. The WRN exonuclease initiates digestion from 3'-recessed termini, and will initiate degradation at a duplex blunt end if the substrate also contains a junction or alternate structure, such as a bubble, forked duplex, and a Holliday junction (12). The exonuclease was also found to degrade the DNA strand of an RNA–DNA heteroduplex (13). Whether the WRN helicase and exonuclease act coordinately in a molecular pathway in vivo is currently unknown. However, we recently demonstrated that the WRN helicase and exonuclease act simultaneously at opposite ends of a DNA forked duplex containing one blunt end (14). The WRN helicase and exonuclease act in concert to remove a DNA strand from a long forked duplex that is not completely unwound by the helicase alone. Digestion at the blunt end shortens the duplex length so that the helicase can fully unwind it. These studies suggest the helicase and exonuclease may act coordinately to remove DNA repair and recombination intermediates.
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Intracellular localization of WRN |
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TopAbstractIntroductionWRN protein biochemistry and...Intracellular localization of...Roles for WRN in...WRN roles in p53-mediated...Function of WRN in...Telomere metabolismPerspectiveReferences |
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The nuclear targeting of the WRN protein is due to the presence of a classical monopartite nuclear localization signal (NLS) in the C-terminal region of the protein [amino acids (aa) 1370–1375] (15) (see Figure 4A). All mutations identified thus far in WS patients result in a truncated WRN protein that lacks the C-terminus and the NLS (reviewed in ref. 16). Thus, the inability of WRN to be transported into the nucleus seems to be critical for the pathogenesis of WS. Once in the nucleus, WRN localizes in the nucleoli (17–20), nuclear foci (20–23) and the nucleoplasm (nuclear diffuse) (17,20) (see Figure 4B). The percentage of WRN molecules located in different nuclear sites is variable and modulated by several factors that will be reviewed briefly.
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The human WRN protein localizes to the nucleoli in a variety of cell types (17), and this localization is modulated by cell cycle and DNA damage. Exponentially growing cells display nucleolar localization of the protein, whereas quiescent (G0), S-phase arrested and damaged [4-nitroquinoline 1-oxide (4-NQO), ultraviolet (UV) light, bleomycin, camptothecin (CPT) and etoposide treated] cells show WRN outside the nucleoli (18,21,24,25). Furthermore, WRN leaves the nucleoli upon inhibition of rRNA transcription following actinomycin-D treatment (26). As transcription arrests in response to some types of DNA damage, it will be important to determine whether WRN nucleolar translocation is induced by the damage per se or by transcription inhibition.
Post-translational modifications of WRN are also directly implicated in the nucleolar localization of the protein. Recent work indicates that acetylation of WRN by p300 results in translocation of the protein to nuclear foci (24). It was proposed that tyrosine phosphorylation, either by modification of WRN directly or of a putative ‘WRN-nucleolar carrier’, may modulate the nucleolar trafficking of WRN (18). A possible candidate for such a carrier is the AAA ATPase VCP/p97, which co-localizes with WRN in the nucleolus and reciprocally co-immunoprecipitates with WRN (F.E.Indig, personal communication). VCP/p97 is tyrosine phosphorylated at its C-terminal domain, and hydrogen peroxide treatment greatly increases VCP tyrosine phosphorylation (27). WRN is phosphorylated at serine/threonine residues, and is also modified by SUMO-1 (28–30). The consequences of these modifications in the transport of WRN into and out of the nucleoli remain to be elucidated. Interestingly, the mouse homolog of the protein (mWRN) does not localize to the nucleoli and displays a nuclear diffuse staining pattern. The difference in the nucleolar localization of WRN between human and mouse cells may partially explain the lack of accelerated aging phenotype in mWrn knockout mice (31).
What targets WRN to the nucleolus? WRN contains a nucleolar targeting sequence (NTS) in the C-terminal domain of the protein (19,20,32). Suzuki et al. (32) proposed that two basic C-terminal aa (1403–1404) represent the WRN NTS. Our laboratory, using a battery of N and C-terminal WRN fragments fused to EGFP, has mapped the WRN NTS to a 144 aa region (aa 949–1092) of the RecQ conserved (RQC) domain. This NTS is dependent on the presence of an active NLS, indicating that the nucleolar targeting process is directly linked to the nuclear import process (33). The discrepancy in the location of the WRN NTS region may be explained by differences in experimental methods. In our studies nucleolar localization was visualized in living cells (unfixed) (20), whereas Suzuki et al. analyzed fixed cells (32). Fixation processes have been found to alter the subcellular localization of proteins. We have observed that different fixation protocols greatly influence the WRN nucleolar localization (C.von Kobbe and V.A.Bohr, unpublished results), thus, emphasizing the importance of verifying protein localization in living cells.
The role of WRN in the nucleoli is still unclear. WRN may localize in nucleoli to participate in nucleolar processes such as transcription and/or for temporal storage (sequestration). A putative partner for nucleolar sequestration of WRN is VCP/p97, which co-localizes with WRN in undamaged, but not CPT-treated cells (F.E.Indig, personal communication). Nucleolar sequestration may serve to regulate WRN activity and WRN physical and functional interactions with protein partners in the nucleoplasm. However, some data suggest that WRN plays an active role in the nucleoli. A recent study found that WRN co-immunoprecipitates with a subunit of the RNA polymerase I complex, and that WS fibroblasts display a deficiency in rRNA levels that can be rescued by expression of exogenous WRN (26). On the other hand, while we reported a deficiency in RNA polymerase II-mediated transcription, we did not detect a deficiency in RNA polymerase I transcript levels (34). However, we have observed that methylation of rRNA genes is accelerated in WS fibroblasts (35). Thus, the role of WRN in the nucleoli may involve active roles in rDNA, rDNA/rRNA and/or rRNA metabolic processes.
The number of WRN containing nuclear foci is dependent on the cell type, DNA damage and cell cycle phase. When present in nuclear foci, WRN partially co-localizes with promyelocytic leukemia (PML) bodies, BLM, TRF2, replication protein A (RPA) and Rad51 (21,23,33,36). The partial co-localization of WRN with these protein partners indicates that the formation of protein complexes (foci) is a dynamic process triggered by different (internal and/or external) stimuli. In telomerase-negative immortalized cells the number of WRN containing nuclear foci is higher than in telomerase positive cells (33). The immortalized telomerase-negative cells contain a unique type of PML bodies (ALT-associated PML bodies; AA-PML) that includes the proteins PML, RPA, Rad51, Rad52, TRF1 and TRF2 (37). AA-PML bodies have been proposed to be sites of telomere lengthening via recombination (see later). The number of WRN containing nuclear foci also increases after replication fork arrest (early S-phase) and upon DNA damage (4-NQO, UV, bleomycin, CPT and etoposide treatment). In both circumstances, WRN leaves the nucleoli and co-localizes with RPA and Rad51 in nuclear foci (21,22). Thus, WRN appears to localize to nuclear regions where transcription is highly active (nucleolus), replication takes place (replication forks), DNA repair occurs (DNA damage ‘foci’), as well as potential sites of telomere metabolism (AA-PML bodies).
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Roles for WRN in DNA replication |
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Several lines of cellular and biochemical evidence support a role for WRN in DNA replication. Specifically, cells derived from WS patients undergo premature replicative senescence (38,39), display an extended S-phase (40) and show a reduced frequency of replication initiation sites (41,42), compared with normal cells. Furthermore, the Xenopus laevis WRN homolog FFA-1 co-localizes with, and is required for the assembly of, RPA containing replication foci (43). Consistent with this, WRN has been found to physically and functionally interact with RPA, and with other enzymes that are critical for DNA replication (Table I). RPA is required for replication (44), and enables the WRN helicase to unwind long duplexes up to nearly 1000 bp (45). In addition, WRN co-localizes with RPA upon replication fork arrest after treatment of cells with hydroxyurea (22). Consistent with a role in DNA replication, WRN also interacts with topoisomerase I (topo I) and proliferating cell nuclear antigen (PCNA) (13,46). In addition, WS cells are hypersensitive to the topo I inhibitor CPT (47,48). Therefore, WRN may act with topo I during replication to resolve blocks due to topological problems in the DNA. WRN may specifically function at replication fork blocks; either to resolve the block and/or to participate in the replication restart process. Biochemically, the WRN helicase unwinds replication fork structures very efficiently (49,50), further supporting a role in replication.
WRN also physically and functionally interacts with a primary DNA replicative polymerase, DNA polymerase 
(pol ). WRN was found to bind to the p50 subunit of DNA pol by yeast two-hybrid analysis, and to associate with the pol dimeric active core (p50 and p125 subunits) by immunoprecipitation (19). Expression of exogenous WRN resulted in the translocation of the p50 subunit to the nucleolus, suggesting a mechanism for WRN regulation of pol (19). In addition, WRN increases the rate of nucleotide incorporation by yeast pol on a primer/template substrate, but this effect is not observed when the processivity factor PCNA is also present (51). WRN was not found to alter the nucleotide incorporation rate of DNA polymerases 
, ß or 
using a primer/template substrate (51). WRN also enables Pol to synthesize past hairpin and tetraplex structures of the d(CGG)n trinucleotide repeat sequence, in a manner dependent on the helicase activity of WRN (52). Consistent with this, the WRN helicase has demonstrated the ability to unwind triplex and tetraplex DNA (49,53,54). The collective evidence suggests that the interaction between WRN and pol may function to alleviate blocks to DNA synthesis that may occur not only during DNA replication, but also during DNA repair and recombination events.
A physical and functional interaction between WRN and the structure-specific endonuclease 1 (FEN-1) also implicates WRN in the processing of Okazaki fragments during lagging strand DNA synthesis. Evidence in yeast supports an important role for FEN-1 in the processing of Okazaki fragments (55). WRN was found to interact with FEN-1 by co-immunoprecipitation, and to stimulate the flap cleavage activity of FEN-1 in vitro, in a manner that is independent of WRN catalytic activities (56). Thus, WRN may function to stimulate FEN-1 cleavage of displaced flaps that occur during lagging strand DNA synthesis at the Okazaki fragments. Consistent with this, WRN was observed in vitro to stimulate cleavage by FEN-1 of several intermediates that may arise during Okazaki fragment processing (57). Defects in Okazaki fragment processing could lead to stalling of the replication fork complex and genomic instability. However, it is important to note that the enzymes mentioned above are also important for DNA repair processes, particularly long patch base excision repair, and their interaction with WRN may also be important for DNA repair and/or recombination (see later).
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WRN roles in p53-mediated pathways |
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A role for WRN in DNA damage response pathways is supported by a physical and functional interaction with the important tumor suppressor protein p53. The p53 protein is a critical regulator of cell cycle arrest, apoptosis and senescence (for review see ref. 58). The ability of p53 to regulate WRN expression levels is supported by the finding that overexpression of p53 results in a decrease in Sp-1-mediated transcription of the WRN gene (59). In addition, WRN has been found to interact with p53 both by co-immunoprecipitation, as well as in vitro with purified proteins (60,61). Identified examples of functional interactions between WRN and p53 include; (i) overexpression of WRN leads to increased p53-dependent transcriptional activity (60); (ii) p53-mediated apoptosis is attenuated in WS cells (61); and (iii) the WRN exonuclease is inhibited by wild-type p53, while two naturally occurring p53 mutants display reduced inhibition of WRN exonuclease activity (25). Furthermore, WRN translocates to nuclear foci upon S-phase arrest induced by hydroxyurea, and subsequently co-localizes with p53 (25), suggesting that these two proteins may act together in a cellular response to replication fork arrest. When a mouse Wrn knock out strain, which displays no obvious phenotype, was cross-bred with p53-null mice, an increased rate in mortality was observed in the resulting double knock out (31,62). Introduction of a p53-null mutation in a different Wrn knock out strain results in an accelerated rate of tumor development and increased variability in the neoplasm types, compared with the p53-null mouse (62). Collectively, the data support a function for the WRN and p53 interaction in a DNA damage response pathway that is important for maintaining genomic integrity. However, much work is still required to define the precise molecular pathways in which they cooperate.
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Function of WRN in DNA repair pathways |
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Several lines of evidence indicate that the WRN protein may play a role in DNA repair. WS cells are characterized by non-homologous chromosome exchanges, termed variegated translocation mosaicism and large chromosomal deletions (63,64) consistent with possible defects in DNA repair. Chromosomal rearrangements can result from loss in the fidelity of repairing DNA double strand breaks (DSB), which are repaired either by non-homologous end joining (NHEJ) or homologous recombination (HR). In addition, sensitivity to selective DNA-damaging agents, and interaction with several proteins involved in DNA repair, support a role for WRN in specific DNA repair pathways that will be reviewed in the following section.
Non-homologous end joining
Three independent laboratories have reported a physical and functional interaction between the WRN protein and two critical components of the NHEJ pathway for repairing DSBs, namely the Ku heterodimer (Ku70, Ku86) and the DNA-dependent protein kinase (DNA–PK) complex. DNA–PK is a serine/threonine kinase that consists of a large catalytic subunit (DNA–PKcs) and the Ku heterodimer bound to DNA. The kinase activity of DNA–PKcs is activated upon association with Ku and DNA. Deficiencies in either component causes hypersensitivity to ionizing radiation (IR), due to defects in NHEJ-mediated repair of the resulting DSBs (reviewed in ref. 65). Two independent studies have observed that SV-40 transformed and hTERT WS fibroblasts display a mild, but significant, sensitivity to IR, compared with the appropriate control fibroblasts (66) and WS fibroblasts expressing exogenous WRN (29). These findings suggest that WRN may participate in DSB repair with its interacting partners Ku and the DNA–PK complex.
The Ku heterodimer was originally identified as the most prominent binder to a C-terminal fragment of WRN, in HeLa nuclear extract preparations (67). Our lab and the Comai lab have also found that Ku interacts with the N-terminus of WRN, where the WRN exonuclease domain resides (68,69). In addition to confirming the physical interaction, several independent studies have indicated that Ku strongly stimulates the WRN 3' to 5' exonuclease not only by increasing WRN's processivity (30,67), but also by expanding the spectrum of substrates that can be degraded by the WRN exonuclease (68). Specifically, the presence of Ku stimulates the WRN exonuclease to efficiently degrade ssDNA and blunt-ended duplex DNA (70), and to bypass some DNA adducts that are normally poor substrates for WRN alone (71). The data suggest a role for Ku in stimulating the WRN exonuclease, perhaps in the processing of DNA ends.
In contrast to the WRN–Ku interaction, reports from various laboratories describing a physical and functional interaction between WRN and the DNA–PK complex are more discrepant. Our observation that Ku mediates a physical interaction between WRN and the DNA–PK catalytic component, DNA–PKcs (30), was confirmed by studies in the Comai lab (72). However, the Chen lab has reported a direct interaction between WRN and DNA–PKcs using purified proteins (29). In addition, our lab and the Chen lab have each observed DNA–PK-dependent phosphorylation of WRN both in vitro with purified proteins, and in vivo using various cell lines (29,30). Specifically, we reported DNA–PK-dependent phosphorylation of WRN after treatment of cells with the radiomimetic drug bleomycin and the carcinogen 4-NQO (30). WS cells are hypersensitive to 4-NQO (73). As DNA–PKcs is activated upon association with Ku bound to DNA, and DNA–PK-dependent phosphorylation of WRN occurs in vitro and in vivo, one could argue that WRN interacts with the DNA–PKcs·Ku·DNA complex, at least transiently, in vivo. Consistent with this, both our lab and the Chen lab observed that WRN, Ku and DNA–PKcs form a complex on DNA by gel shift analysis (29,30). In contrast, the Comai lab reported that addition of WRN resulted in dissociation of DNA–PKcs from Ku and the DNA (72). The reasons for the discrepancies are unknown, but may be ascribed to differences in purified protein preparations and/or experimental conditions. Nevertheless, WRN appears to be a substrate for DNA–PK phosphorylation both in vivo and in vitro.
The consequences of DNA–PK complex associating with and phosphorylating WRN, on the WRN catalytic activities, suggest a regulatory role for DNA–PK. Data from our lab and the Chen lab agree that the WRN exonuclease and helicase activities are negatively modulated by DNA–PKcs. However, the precise mechanism of inhibition differs. We observed inhibition of WRN exonuclease and helicase via phosphorylation by activated DNA–PKcs complexed with Ku and DNA (30), whereas, the Chen lab reported WRN helicase and exonuclease inhibition via a physical interaction with DNA–PKcs alone (29). While the Comai lab did not detect an effect of DNA–PK on WRN exonuclease, their experiments included a pre-incubation step of DNA–PKcs and Ku with the DNA substrate, prior to adding WRN (72). Autophosphorylation of the DNA–PK complex, which could have occurred during this pre-incubation, weakens the kinase activity, and results in dissociation of DNA–PKcs and Ku (74). Thus, differences in the phosphorylation state of DNA–PK among the studies may account for some of the discrepancies.
Current models suggest that phosphorylation of WRN by DNA–PK may function to negatively regulate WRN end processing during NHEJ (Figure 5). In this model, DNA DSB repair is initiated by Ku binding to the broken DNA ends, bringing them into close proximity. The ends are then processed by a combination of DNA unwinding and/or degradation, perhaps by WRN, to reveal sites of microhomology that then anneal. The WRN exonuclease, and WRN stimulation of FEN1 flap cleavage (56), may serve to remove the resulting flaps and to tidy up the ends for proper ligation. Presumably, DNA–PKcs is recruited by Ku, and the activated DNA–PK complex may limit the extent of end degradation by negatively regulating the WRN exonuclease, thereby preventing extensive deletions. In the absence of WRN, other more promiscuous nucleases may substitute. Consistent with this model, in a cellular assay that measures NHEJ with a linear plasmid, WS cells displayed extensive deletions at the non-homologous joined ends, compared with normal cells (75).
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Recombinational repair and recombination
Biochemical and genetic evidence strongly suggest the importance of the homologous recombinational (HR) repair pathway in maintaining chromosome stability and in repairing DSBs. Cellular DNA recombination can occur physiologically during meiotic DNA replication and V(D)J recombination, or can be induced by DNA-damaging agents including IR and DNA cross-linking agents. Cells from WS patients are hypersensitive to DNA cross-linkers and, to a lesser extent, IR (29,76). WS cells also display reduced repair of DNA psoralen cross-links (77). Stalled replication forks can also lead to DSBs and/or recombination intermediates that must be resolved to re-initiate replication (for review see ref. 78). Evidence suggests WRN may function at stalled replication forks (see earlier). Studies in Escherichia coli suggest that RecQ functions with the 5' to 3' exonuclease RecJ at replication forks blocked by UV-induced DNA damage (79). Also, RecQ suppresses illegitimate recombination in E.coli through initiating homologous recombination and disrupting joint molecules (80). In yeast Saccharomyces cerevisiae, defects in the RecQ homolog Sgs1 leads to hyper-recombination (81), which can be suppressed by overexpressing human WRN (82). However, much debate has surrounded the potential role of WRN in recombinational repair until recent reports from Monnat et al. (66,83), which strongly suggest that WRN functions in resolving recombination intermediates in Rad51-dependent HR (see later).
Some proteins that participate in the recombinational repair pathway have been found to functionally interact with WRN. Increasing evidence suggests that the BS protein, BLM, functions in HR and at stalled replication forks (for review see ref. 84). Cells from BS patients show increased sister-chromatid exchange, a hallmark of DNA recombination (reviewed in ref. 85). We recently reported that BLM interacts with WRN by co-immunoprecipitation and co-localization, and that the purified proteins interact in vitro (33). While BLM inhibits the WRN exonuclease activity, co-operativity between their helicase activities using various substrates has not yet been identified (33). In light of the similarities between the WRN and BLM helicase activities and substrate specificities in vitro, including recombinational intermediates (reviewed in ref. 86), it is possible they function in a synergistic manner to cope with DNA damage. Synergistically increased hypersensitivities to genotoxic agents including 4-NQO, CPT, methyl methanesulfonate (MMS) and UV, were observed in WRN-/-/BLM-/- chicken cell lines, compared with the single knock out cells. The data suggest that WRN and BLM may function in DNA repair pathways, including HR, in a complementary manner and may possess redundant, partially overlapping functions. However, it has also been suggested that WRN and BLM may act primarily at different steps in the recombinational pathways, based on differences in cellular and clinical phenotypes between WS and BS (for review see ref. 87). Future experiments are required to examine potential WRN and BLM interaction and cooperation in cells after DNA damage, particularly during DSB repair.
The recombinational repair Rad51 paralogs include Rad51, Rad51B, Rad51C, Rad51D, XRCC2 and XRCC3. Rad51 plays a central role, and other Rad51 paralogs function in assisting Rad51 during recombinational repair (88). A recent report shows that WRN co-localizes with Rad51 and RPA in response to CPT treatment (21). Rad51 functions together with RPA to coat and stabilize single-strand DNA for a strand exchange activity, an early step in recombination shortly after the single strand DNA appears. Both WRN and BLM interact with RPA (45,89), and BLM has been found to interact with Rad51 both in vitro and in vivo (90). Recent studies from Monnat et al. (83) indicate that WS cells display a >20-fold reduction in viable mitotic recombinants using a neo gene selection assay. However, recombination initiation and rates appeared normal in WS cells in the absence of cell division, with a LacZ expression assay (83). Remarkably, the generation of viable neo+ recombinant progeny was rescued by the expression of WRN or the bacterial resolvase protein RusA, which also improved WS cell survival after DNA damage (66). The data suggest a deficiency in resolving recombination intermediates in the absence of functional WRN. In addition, the suppression of Rad51-dependent recombination, by expressing a dominant-negative Rad51 protein, significantly reversed the DNA damage hypersensitivity in WS cells (66). The data suggest that a deficiency in resolving DNA recombination intermediates may contribute to the DNA damage hypersensitivity, limited cell growth and genomic instability phenotypes observed in WS cells. WRN may mediate the resolution of recombinational intermediates in association with Rad51, RPA, BLM and possibly as yet unidentified interactors. Very recently, we have found that WRN interacts physically and functionally with the Mre11·Rad50·Nbs1 complex (W.-H.Cheng et al., submitted for publication), which functions in HR. We are currently exploring the mechanism of this interaction and trying to delineate the cellular pathway. Understanding the roles of WRN in recombinational repair is at an exciting but early stage.
Base excision repair
Some evidence suggests WRN may play a specialized role in base excision repair (BER), which is an important pathway in dealing with some of the most common DNA lesions including alkylated, oxidized or deaminated bases. In the BER process, the abasic intermediate is subsequently cleaved to generate a nick with a 3'OH and a 5'-deoxyribose 5-phosphate (dRP). In short patch BER, DNA pol ß incorporates a single nucleotide and removes the 5'dRP. If the dRP is modified and refractory to excision by pol ß, long patch BER occurs in which 2–8 nucleotides are incorporated by pol ß and/or pol , and the resulting 5'dRP containing flap is cleaved by FEN-1 (reviewed in ref. 91). Both pol ß-/- mouse cells and WRN-/- mutant chicken cells show hypersensitivity to MMS, an agent which produces DNA damage that is repaired via BER, compared with the isogenic wild-type cells (92,93). However, repair of apurinic/apyrimidinic (AP) sites by whole cell extracts from WS cells appears to be normal (77), indicating that WRN does not likely function normally in short patch BER. Rather, WRN probably participates in specialized BER processes including long patch BER. The substrates on which the WRN helicase and exonuclease act, as well as the proteins with which WRN interacts, strongly support this hypothesis.
WRN has been shown to interact physically and/or functionally with several replication proteins which also participate in long patch BER including pol , PCNA, RPA and FEN-1 (see Table I). The ability of WRN to stimulate pol to synthesize past hairpin and tetraplex structures of the d(CGG)n trinucleotide repeat sequence (52) may be important for repair initiated in genomic sequences susceptible to alternate structure formation including telomeric DNA. WRN also interacts with two proteins found to stimulate long patch BER, namely PCNA and RPA. While WRN interacts physically with PCNA (13,46), a functional interaction has yet to be determined. The ability of RPA to stimulate the WRN helicase unwinding of long substrates (45,94) may assist WRN unwinding of BER intermediates (see later). FEN-1 performs the critical role of cleaving the flap generated in long patch BER, a step which WRN may stimulate as WRN demonstrates the ability to stimulate FEN-1 activity on various substrates (56,57). Deficiencies in the removal of the 5'dRP containing flap could potentially lead to genomic instability. Collectively, the data suggest that WRN may participate in long patch BER by unwinding DNA intermediates and/or by stimulating FEN-1 flap cleavage.
We have recently identified a physical and functional interaction between WRN and pol ß (94a). An active WRN helicase domain stimulates pol ß strand displacement DNA synthesis at a nick on a BER substrate. WRN stimulation of pol ß nucleotide incorporation was not observed on a primer/template substrate, which lacks a downstream oligonucleotide, as reported previously (51). Therefore, WRN stimulation of pol ß is substrate dependent, and thus, differs from the previously reported WRN stimulation of pol (see section Roles for WRN in DNA replication). Furthermore, WRN can unwind a BER substrate produced following uracil-DNA glycosylase and AP endonuclease (APE1) treatment of a uracil-containing oligonucleotide (94a). Together, these results provide further evidence for a role of WRN in BER.
The weak strand displacement activity of pol ß results in the expansion of CAG/CTG triplet repeats (95). Weak strand displacement activity during DNA repair at strand breaks may enable short tracts of repeat sequences to be converted into longer, more mutable stretches associated with neurological diseases. As polymerase-initiated DNA synthesis errors most probably play a central role in human aging and disease, it is interesting to speculate that WRN may increase the fidelity of polymerases (such as pol ß and pol ) via unwinding of alternate structures and/or through stimulation of strand displacement DNA synthesis (pol ß). Also, it will be interesting to test if WRN can relieve blocks to pol ß DNA synthesis at sites of alternate DNA structures, including tetraplex DNA, as observed for pol (52). Specialized roles for WRN in BER may depend on the genomic sequence in which repair is initiated.
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Telomere metabolism |
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Some of the WS cellular phenotypes, including genetic instability and a decline in proliferative competence, are consistent with defects in telomere metabolism. Telomere shortening and dysfunction cause replicative senescence and genomic instability; however, telomere-associated senescence can be bypassed by the expression of telomerase, which extends telomeres (for review see ref. 58). Three independent studies reported that expression of exogenous telomerase in WS fibroblasts extends the cellular life span, suggesting that premature senescence in WS cells may be related to telomere dysfunction (96–98). Furthermore, the expression of exogenous telomerase also partially reverses the hypersensitivity to 4-NQO displayed by WS cells (99). The decrease in proliferative capacity of WS cells cannot be explained simply by acceleration of telomeric loss. Although WS fibroblasts demonstrate accelerated rates of telomere shortening, at senescence, the mean telomere lengths are longer in WS cells compared with those of the senescent controls (100,101). However, recent reports indicate that alterations in telomere structure, rather than telomere length, trigger replicative senescence (102,103). Therefore, deficiency in WRN may lead to disruptions in telomere integrity and/or structure that could be complemented by protective effects exerted by telomerase.
WRN protein interactions also suggest that WRN may function in telomere metabolism. The physiological function of the WRN·Ku, WRN·DNA–PK and WRN·Mre11 complex interactions may be important not only for NHEJ, but also for telomere maintenance. Several enzymes involved in DSB repair, including the Ku heterodimer and DNA–PKcs (104,105), as well as the Mre11·Rad50·Nbs1 complex (106), have been found to localize to telomeres (Figure 6). Defects in these enzymes result in dysfunctional telomeres, manifested as accelerated telomere shortening and/or telomere end fusions (107–110). How these repair proteins function in telomere maintenance is still not well understood, but they may act in a cellular response to dysfunctional and/or damaged telomeres. Furthermore, Ku interacts with the critical telomere binding proteins TRF1 and TRF2 (111,112), and Nbs1 interacts with TRF2 (113). Both TRF1 and TRF2 regulate telomere length (114), and defects in TRF2 induce telomere end fusions and either growth arrest or p53-mediated apoptosis (102,115,116). We have recently reported that the WRN protein also interacts with TRF2 by co-immunoprecipitation and co-localization, and that the purified proteins interact in vitro (36). In addition, we found that TRF2 also interacts with RecQ family member, the BLM protein, and promotes DNA unwinding by the WRN and BLM helicases in vitro (36). Consistent with this, BLM was found to partially co-localize with telomeric DNA (117). Collectively, these studies suggest that WRN may function in a protein complex at telomeres, in cellular pathways that impact telomere integrity, and possibly respond to dysfunctional telomeres.
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How might WRN function with its protein partners at telomeric ends? Electron microscopy studies have shown that telomeres in mammalian cells form t-loop structures in which the single-stranded 3' tail invades the homologous duplex telomere region creating a displacement loop (D-loop) (118) (Figure 6). Consequently, the telomeric end is protected and sequestered, thus, preventing its recognition as a DSB. The formation of telomeric t-loops in vitro requires TRF2, is promoted by the presence of p53, and is predicted to be stabilized by either a D-loop or a Holliday junction (119,120). Presumably, structures at telomeric ends must be resolved for telomerase, the DNA replication forks, and DNA repair proteins to gain access to the terminal region of the telomere. The WRN and BLM proteins are probable candidates to participate in this process, as they unwind various DNA secondary structures including Holliday junctions (22,49), G4-quartets which can form in the telomeric G-rich sequence (49), as well as D-loop structures (121,122). Furthermore, p53 not only interacts with both WRN (Table I) and BLM (123), but also regulates the WRN and BLM helicases on Holliday junctions (124), and the WRN exonuclease (25), and could potentially regulate their activity at telomeric ends. As the BLM helicase is expressed primarily during the S/G2-phase, it may function specifically in resolution of telomeres during DNA replication with WRN and TRF2.
Potential roles for the WRN helicase and its interacting partners in recombination at telomeres are derived from studies in yeast. Three independent studies reported that the RecQ homolog in S.cerevisiae, Sgs1p, participates in a telomerase-independent pathway for telomere lengthening (23,125,126). Expression of the mouse Wrn homolog partially rescues this defect in the telomerase-negative, sgs1 mutant strain (125). This mechanism, termed alternative lengthening of telomeres (ALT), is predicted to involve recombination as some recombination enzymes are required (reviewed in ref. 127). Evidence for a similar pathway has been found in telomerase-negative immortalized mammalian cells (128). These cells are characterized by long heterogenous telomeres and distinct nuclear foci referred to as ALT-associated PML bodies, which contain Rad52, Rad51, RPA, TRF1, TRF2, telomeric DNA (37), Ku (105) and Nbs1 (106). Both WRN and BLM were found to localize in AA-PML bodies (23,36,117). Notably, WRN and BLM interact physically and/or functionally with many of the proteins in these foci, suggesting that these RecQ helicases may act in a protein complex at telomeric ends in recombination pathways. Future work is required to determine the precise roles for WRN at telomeres in both normal and ALT mammalian cell lines.
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Perspective |
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The increasing number of WRN interacting proteins argues for a putative role for WRN in multiple DNA metabolic processes (Figure 7). In this scenario, the multi-functional roles of WRN could explain its complex and dynamic intranuclear localization. Clearly the status of WRN localization in the cell may determine which protein complexes and pathways WRN will be engaged in. Therefore, understanding the regulation of WRN localization in the cell will be critical for understanding its cellular roles. Alterations in the WRN protein in response to several internal (cell cycle, telomeric ‘state’, endogenous DNA damage) and/or external (exogenous DNA damage) stimuli, may determine the targeting of WRN to different intranuclear structures. These alterations may include post-translational modifications and/or interactions with other proteins. Analysis of the shuttling of WRN in living cells in response to various stimuli will be important to address these issues.
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It appears that the central function of WRN is in DNA repair. WRN participates in several DNA repair pathways, and while it may not be essential in any individual process, it does appear to have significant functional roles. WRN probably acts to resolve DNA intermediates that arise normally as a result of DNA repair processes, and/or that result from complications encountered during DNA repair and replication (i.e. DNA triplex formation). In recent years, a number of DNA repair proteins have turned out to be involved in more than one DNA repair pathway, which is of great interest in the field. It is possible that WRN coordinates or regulates transition between different pathways as they progress after DNA damage. This regulation or coordination may be affected through various post-translational modifications of the WRN protein or its partners, including phosphorylation, acetylation, sumoylation or oxidation. Phosphorylation, for example, regulates WRN catalytic activities and may also affect the binding to and interactions with its partners. Thus, much work is still required to understand the regulation of WRN participation in various protein complexes and DNA metabolic pathways.
Despite significant advances in our understanding of the WRN protein and WS, important questions continue to challenge investigators in the field. For example, the relative physiological importance of WRN participation in various protein complexes and cellular pathways, especially in maintaining genomic integrity, remains to be elucidated. Also unknown is how the lack of WRN helicase and exonuclease activities, and the absence of WRN protein interactions, contribute to the plethora of premature aging symptoms in WS. Recent progress has laid the foundation for addressing these important questions, and should lead to new insights regarding normal aging and cancer progression.
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