Function Of

Identification of proteins that interact with WRN has helped to shed light on the in vivo functions of WRNp. Some laboratories have characterized the association of

WRN with specific candidate interacting proteins, whereas others have screened cDNA libraries by the yeast two-hybrid system, isolated the WRN complex from the cell extracts, or captured the interacting proteins by binding to a WRNp affinity column. Interestingly, each method identified different sets of WRN-interact-ing proteins. Thus, WRN may associate with different protein complexes depending on the status of DNA metabolism during the time that the WRN protein is recruited.

Cell biological studies also suggest that WRN has multiple functions. Two characteristics of WS cells that had been well known even before the WRN gene was identified are that these cells exhibit a shortened replicative life span and ge-nomic instability. More recently, studies of drug sensitivities and telomeric metabolism led to a better understanding of the specific functions of WRNp (discussed in more detail in Sec. III.E). The identification of proteins interacting with WRNp along with results of cellular and subnuclear studies suggest that WRN may be involved in a wide variety of DNA metabolic processes.

A. DNA Replication

FFA-1 is a Xenopus ortholog of WRN and was originally identified in the replication initiation complex of Xenopus oocytes as being required for the formation of replication foci during DNA replication (42). FFA-1 and the p70 subunit of RPA also have been coimmunoprecipitated from lysates of Xenopus oocytes (44). The immunodepletion of FFA-1 from total interphase egg extracts has no significant effect on DNA replication. However, RPA stimulates the DNA helicase activity of FFA-1, and this stimulation can be prevented in a dominant negative manner by the addition of glutathione S-transferase FFA-1 fusion proteins. Therefore, it remains to be determined how essential FFA-1 is for DNA replication, and to what extent other helicases can compensate for their absence (44). WRNp interacts with several components of the DNA replication complex, including proliferating cell nuclear antigen (PCNA) and topoisomerase I (51). The N-terminal region within the exonuclease domain of the WRNp, particularly the region including amino acids 168-246, strongly interacts with the C-terminal portion of PCNA (51), as indicated in Figure 3. The N-terminal region of recombinant WRNp appears to form mostly trimers, as does PCNA, suggesting that the interaction between WRN and PCNA involves quaternary structure (49).

B. DNA Polymerase 6

DNA polymerase 8 (pol8) participates in DNA replication and repair of DNA damage. This enzyme is also found in the telomeric complex. Evidence of physical interaction between WRN and pol8 initially came from a yeast two-hybrid screen, in which the C-terminal region of WRNp was used as the "bait" to capture

Figure 3 Proteins that interact with the WRN protein. Proteins known to interact with WRNp at specific sites are shown schematically. The approximate regions of proteinprotein interaction are indicated.

the p50 subunit of polS, as shown in Figure 3 (52). Immunoprecipitation with an anti-p50 antibody resulted in a complex including p120, the catalytic subunit of polS. In yeast, physical and functional interaction between WRN and polS requires the third subunit pol32p (53).

Functional interaction of WRNp and polS appears to be unilateral. In a simple primer extension assay, the addition of WRNp enhanced polS activity, whereas the addition of polS did not stimulate exonuclease or helicase activities (53). In contrast, WRNp did not stimulate either pola- or pole-mediated DNA synthesis (53, 54). The presence of WRNp also enabled polS to transverse hairpin and G'2 bimolecular tetraplex structures to complete DNA synthesis (54). One of the functions of WRNp may be to recruit polS to regions of complex secondary DNA structure to alleviate stalled DNA synthesis (52,54).

C. Homologous Recombination

One of the important biological functions of E. coli RecQ is to suppress homologous recombination by disrupting its intermediate structure (55). The yeast homolog of RecQ, Sgsl, is involved in homologous recombination and in illegitimate recombination or nonhomologous end joining (NHEJ). The WRN and BLM helicases are able to complement increased homologous recombination and illegitimate recombination of an Sgsl deletion mutant (56). However, the cytogenetic characterization of cells from Bloom syndrome patients show increased exchanges of sister chromatids, whereas cells from patients with WS exhibit variegated translocation mosaicism (57). These findings suggest that the BLM helicase may play a more prominent role than WRNp in the suppression of homologous recombination in vivo.

D. Repair of Breaks in Double-Stranded DNA

DNA-PK is a protein complex including a protein kinase catalytic subunit, DNA-PKcs, and a regulatory subunit, Ku70/80 (58). This protein complex is involved in the initial stages of nonhomologous end joining (NHEJ). WRNp has been shown directly to interact with DNA-PKcs and Ku80 (48,50,58). Association of WRN with the Ku complex enhances its exonuclease activity but has no effect on its helicase activity (50). Upon binding to Ku, WRN is able to degrade DNA from the 5' recessed end (5'^3' exonuclease activity) and from the blunt end. This may explain why both 3'^5' (3,46,60) and 5'^3' (36) exonuclease activities were observed in recombinant WRNp. The presence of exonuclease activity has been speculated to be a necessary step for DNA-PK-mediated NHEJ prior to the polymerization and ligation step mediated by XRCC 4 and ligase IV (58). In addition, the WRN helicase may unwind broken ends of double-stranded DNA in the search for microhomology.

E. Telomeric Maintenance

In a high-quality immunofluorescence study, Shiratori et al. (61) identified nuclear dots suggesting that WRNp is localized on telomeres. Johnson et al. (10) demonstrated WRNp colocalizes with various telomeric components, including TRF1 and TRF2 in six ALT cell lines. ALT cells lack telomerase activity, and the telomeres in these cells are presumably maintained by recombination. Recombinant WRNp did not unwind a G' 2 tetraplex containing a telomeric repeat sequence (45). However, WRNp did appear to unwind up to 23 kb of a polymerase chain reaction (PCR)-generated telomeric repeat sequence to single-stranded DNA, and this process was stabilized by hRPA (62). These findings collectively suggest that WRNp plays an important role in telomeric maintenance.

During the serial passage of primary WS fibroblasts, telomeres became shortened more quickly, and telomeres in cells that have stopped dividing were longer than those in control fibroblasts (63). Telomeres of WS lymphoblastoid cell lines (LCLs) were unstable, and telomere length varied more widely in LCLs from patients with WS as compared with those from normal subjects (64). The telomere length at which LCLs from patients with WS go into crisis also varied widely. It has been suggested that one way in which WRNp may be involved in the ALT pathway is by regulating the number of extrachromosomal telomeric repeats (65). This may explain why the catalytic subunit of human telomerase (hTERT) is able to extend the replicative life span of WS cells indefinitely (66-68). hTERT may be able to circumvent the early halt of cell division in WS cells because the WRNp complex functions in pathways that are distinct from hTERT-dependent telomeric maintenance (65). These findings provide significant insight into the possible mechanism of mesenchymal tumorigenesis in patients with WS, as ALT cell lines are frequently mesenchymal in origin (10).

However, other important factors also may be involved in regulation of telomeric function. Hisama et al. (71) have shown that either WRN or hTERT could complement 4NQO sensitivity in simian virus 40 (SV40) transformed WS fibroblasts. Introduction of hTERT appears to reprogram gene expression (68). WRNp may play an additional role in telomeric maintenance aside from repairing damaged telomeres and immortalizing cells that lack telomerase activity.

As shown in Figure 3, the C-terminal region of WRNp interacts with the p53 tumor suppressor, as these proteins can be coimmunoprecipitated (69,70). In the absence of WRN, p53-mediated apoptosis is attenuated (69). Overexpression of WRN enhances p53-dependent transcriptional activation of p21Waf1 (70) and potentiates p53-mediated apoptosis (70). Synergistic actions of p53 and WRN have been observed in mouse models of WS (72,73) (as discussed in Sec. IV.D).

A yeast two-hybrid screen using mouse WRN as the bait identified Ubc9 and SUMO-1 as WRN-interacting proteins (74). Ubc9 (ubiquitin-conjugating 9) protein is involved in the covalent attachment of SUMO-1 to its target proteins, and SUMO-1 is a ubiquitinlike protein that modifies other cellular proteins, enhancing their stability or modulating their subcellular compartmentation. The N-ter-minal domain (amino acids 272-514) of WRN interacts with Ubc9 and SUMO-1.

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