Sci. Aging Knowl. Environ., 6 August 2003
Vol. 2003, Issue 31, p. pe22
[DOI: 10.1126/sageke.2003.31.pe22]


Diverse Dealings of the Werner Helicase/Nuclease

Wen-Hsing Cheng, and Vilhelm A. Bohr

The authors are in the Laboratory of Molecular Gerontology at the National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA. E-mail: BohrV{at};2003/31/pe22

Key Words: DNA repair • recombination • Werner • progeroid

Werner syndrome (WS) patients display many symptoms of normal aging, including genomic instability, osteoporosis, high rates of cancer, skin thinning, and atherosclerosis. This syndrome is considered to be a model system for segmental progeria (that is, a disease that involves only some features of the aging process). The gene mutated in WS, WRN, encodes a multifunctional nuclear protein that possesses 3'-to-5' helicase, 3'-to-5' exonuclease, and adenosine triphosphatase activities [for a review, see (1, 2)]. WRN contains an exonuclease domain, an acidic region, a helicase domain, a domain containing RecQ C-terminal and nucleolar targeting sequences, a C-terminal helicase-related region, and a nuclear localization sequence (Fig. 1). WRN interacts physically and/or functionally with a wide variety of proteins with known functions (see Fry Review), and these WRN-interacting proteins provide clues to the functions of WRN.

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Fig. 1. The domains of WRN and their functions. This schematic diagram shows the domain structure of the human WRN protein, including the exonuclease domain, the acidic region, the helicase domain, the RecQ C-terminal and nucleolar targeting sequences (RQC/NTS), the C-terminal helicase-related (HRDC) region, and the nuclear localization sequence (NLS). The functions related to these domains are indicated in the figure.

For example, the interaction of WRN with the DNA-dependent protein kinase (DNA-PK) complex (see below), replication protein A (RPA), DNA polymerase {beta}, the Bloom syndrome protein BLM, and flap endonuclease 1 (FEN-1) suggests that WRN is involved in repairing DNA via homologous recombination, nonhomologous end-joining, and base excision repair (1, 2) (see also Shcherbakova Review). Data that support this suggestion come from experiments with cells cultured from WS patients, which are more sensitive to DNA cross-linkers than to any other genotoxic drugs and are defective in resolving recombination intermediates (3, 4). In addition, although WS cells are as efficient as wild-type cells in annealing linearized plasmids, there are extensive deletions near the regions of the ligation ends in these cells (5). One explanation for this result is that other, more promiscuous exonucleases and helicases may accelerate the processing of DNA ends in the absence of WRN. Further, the observations that WRN interacts with DNA polymerase {delta}, telomeric repeat factor 2 (TRF2), and replication protein A suggest that WRN is also likely to be involved in other aspects of DNA metabolism, including DNA replication and telomere maintenance. How one protein can be involved in such diverse processes has been the subject of extensive research. Now, a paper by Chen et al. (6) published in the August 2003 issue of Aging Cell sheds some light on the multifunctional nature of WRN.

WRN Functions Through Many Partner Proteins

Before discussing in detail the mechanism of action of WRN, we must first introduce the other players in the DNA repair process. DNA double-strand breaks (DSBs) are repaired mainly via homologous recombination and nonhomologous end-joining, two processes thought to involve WRN. The unphosphorylated (inactive) ATM protein, whose gene is mutated in patients with ataxia-telangiectasia, a potential progeroid syndrome, senses DSBs and is activated by autophosphorylation (7). The activated version of ATM interacts with other serine/threonine kinases to phosphorylate the histone H2AX (termed {gamma}-H2AX once it is phosphorylated). {gamma}-H2AX and MDC1, a mediator-type protein that is required for the intra-S-phase and G2/M DNA damage checkpoint (8-10), appear at the damaged DNA sites and recruit 53BP1, another DNA damage checkpoint protein that is activated promptly after the formation of DNA DSBs (11). Many other downstream proteins are recruited to the {gamma}-H2AX foci, including the Mre11 complex (a nuclease that is composed of Mre11, Rad50, and Nbs1/Xrs2) (12) and the breast cancer-related protein BRCA1 (13), and together they promote DSB repair by either homologous recombination or nonhomologous end-joining.

The nonhomologous end-joining pathway is operated mainly by the DNA-PK complex, which consists of three subunits: Ku70, Ku80, and a DNA-dependent protein kinase catalytic subunit (DNA-PKcs ) (see also "Twisted Logic: Discoveries tangle Werner syndrome helicase story"). The Ku proteins dramatically stimulate WRN exonuclease activity (14), but WRN catalytic activities are inhibited by phosphorylation by DNA-PKcs (15, 16). These observations imply the existence of a DNA-PKcs-dependent feedback regulation of WRN catalytic activities, which may limit the potential of WRN in processing DNA at broken ends initiated by the Ku protein. In the nonhomologous end-joining pathway, the Ku proteins bind to DNA DSB ends, which is followed by recruitment of WRN and DNA-PKcs. XRCC4 and DNA ligase 4 then seal the ends to finish repairing the DNA.

The more precise homologous recombination pathway uses the undamaged DNA strand to search for a region of DNA that is homologous to the damaged sister chromatid. The single-stranded DNA generated during this process is protected by Rad51 and RPA, two proteins that interact with WRN (17, 18). Rad52 facilitates Rad51 loading onto sites of recombination and also interacts physically and functionally with WRN (19). In addition, phosphorylated DNA-PKcs colocalizes with {gamma}-H2AX foci during the G1 phase of the cell cycle (20).

Taken together, these data suggest that WRN participates in both pathways to repair DNA DSBs in a cell cycle-dependent manner. As summarized in Fig. 2, DNA DSBs are detected by {gamma}-H2AX, which recruits either Ku heterodimers or the Mre11 complex (which checks for breaks in DNA strands) to regions of DNA DSBs. Recruitment of Ku heterodimers and the Mre11 complex to {gamma}-H2AX may destine cells to repair DNA DSBs via nonhomologous end-joining and homologous recombination, respectively. A recent report clearly shows that Nbs1, an essential component of the Mre11 complex, is required for homologous recombination but not for nonhomologous end-joining (21). The observations that WRN interacts with the Ku heterodimers, RPA, Rad51, and Rad52 suggest that WRN has a role in the early stages of these two pathways. However, the observation that WRN also participates in the late stages of homologous recombination (4) suggests the existence of yet-to-be-identified partners of WRN.

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Fig. 2. Is WRN a transducer of the DNA damage response? Recent advances suggest that WRN participates in the repair of DNA double-strand breaks and base oxidative modification. WRN may participate in the three DNA repair pathways: base excision repair (BER), homologous recombination (HR), and nonhomologous end-joining (NHEJ). Proteins that have been shown to interact with WRN are highlighted as italics.

Why does WRN participate in so many facets of DNA metabolism? One possibility is that WRN may function as a transducer of various DNA repair pathways. After activation, transducers amplify the effects of upstream signals by transmitting the signals to a diverse array of downstream effectors. Although it is not clear how WRN and its protein partners interact in response to DNA damage, some of these partners may function with WRN in a common pathway, and some in parallel pathways.

It is also possible that WRN contributes to DNA repair by affecting the functions of its partner proteins. This mode of regulation seems to be important for the repair of oxidized, alkylated, deaminated, and hydrolyzed bases, the types of DNA damage repaired by the base excision repair (BER) pathway. The sequential actions of several BER proteins recognize and incise the damaged base, which is followed by the actions of specific endonucleases and polymerases that complete the repair process. WRN stimulates FEN-1 cleavage activity and polymerase {beta} strand displacement during DNA synthesis (22, 23), two functions that are important for repairing DNA by the BER pathway. In addition, WRN has been shown to stimulate the Rad52 DNA strand-annealing activity (19). Because WRN-deficient cells are hypersensitive to some agents that generate oxidative stress, WRN may be required to coordinate interactions between base excision repair proteins, to facilitate the stability of the recombination intermediates, and/or to stimulate FEN-1 cleavage of the DNA flap that is produced after the alignment of processed single-stranded ends.

WRN As a Structural Protein

The recent paper by Chen et al. (6) provides yet another plausible explanation for why WRN interacts with so many different proteins: WRN may function as a structural protein that provides a scaffold on which the various reactions could take place. Two experimental results give rise to this hypothesis. First, a mutated version of the WRN protein that is defective in both the helicase and exonuclease activities complements the defect in DSB repair by nonhomologous end joining in WS cells even more efficiently than does wild-type WRN. This altered version of the protein was generated by introducing two point mutations: one in the helicase domain and the other in the exonuclease domain. Second, singly mutated versions of WRN that lacked either the helicase or the exonuclease activity only partially complemented the nonhomologous end-joining defect of the WS cells. The implication of these results is that the two activities of the WRN protein, exonuclease and helicase, must be balanced for the protein to promote appropriate DSB repair. The two WRN catalytic activities can function simultaneously, and unwinding DNA without appropriate end-processing or digesting DNA without strand separation might elicit adverse cellular responses, such as aberrant DNA repair. This hypothesis implies that WRN catalytic activities do not play necessary roles in repairing DNA via the nonhomologous end-joining pathway. Rather, it affects this process by another mechanism, which the authors hypothesize involves the participation of WRN as a structural scaffold that regulates interactions among other proteins (such as proteins involved in DNA repair). As mentioned by Chen et al. (6), the implied structural role for WRN makes sense when we consider the consequences of WRN mutations found in WS patients: A majority of the mutations lead to a loss of WRN nuclear localization. Although it is not known whether the point mutations in WRN that were studied by Chen et al. affect WRN's ability to bind to its partners, these results support the hypothesis that WRN serves as a transducer of DNA repair by interacting with many proteins in response to DNA damage. Understanding the mechanism by which WRN participates in DNA repair may provide insight into the genomic instability and accelerated aging seen in patients with WS.

It is interesting that Chen et al. (6) found that WRN and BLM (another RecQ helicase protein that is defective in Bloom syndrome) repair DNA DSBs in different ways. Their data suggest that WRN and BLM play relatively important roles in repairing DNA DSBs via nonhomologous end-joining and homologous recombination, respectively. The BLM results are in agreement with the fact that cells from Bloom syndrome patients or BLM-deficient mice show increased DNA recombination (24). However, caution should be taken when interpreting these results for WRN, if one considers evidence for WRN in the process of homologous recombination using another assay (4). The nonhomologous end-joining pathway is the dominant one in the G1 phase, whereas the homologous recombination pathway is dominant in S phase. Therefore, WRN may participate in the repair of DNA DSBs via different pathways in different phases of the cell cycle.

Aging is an inevitable biological process, but it is not a disease. In fact, mutations in the WRN protein do not pose an immediate threat to human life; rather, they accelerate the process of aging. The facts that WRN interacts with many proteins that participate in DNA metabolism and that cells from WS individuals show a greater extent of genomic instability than do cells from normal individuals support the hypothesis that a decline in DNA repair capacity contributes to accelerated aging. Current data from experiments with both cultured cells and whole organisms support the view of WRN as an "anti-aging" protein. However, a more detailed description of the mechanisms of WRN actions at the molecular level is required for us to better understand the aging process.

August 6, 2003
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Citation: W.-H. Cheng, V. A. Bohr, Diverse Dealings of the Werner Helicase/Nuclease. Sci. SAGE KE 2003, pe22 (6 August 2003);2003/31/pe22

Werner Syndrome Protein--Unwinding Function to Explain Disease.
R. J. Monnat Jr. and Y. Saintigny (2004)
Sci. Aging Knowl. Environ. 2004, re3
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