In this study we have mapped the physical and functional interactions between the Ku heterodimer and WRN and shown that WRN physically associates with each of the two distinct subunits of the Ku heterodimer. An N-terminal fragment of WRN interacts with Ku70, while a C-terminal fragment of WRN interacts with Ku80. This is shown using two different approaches: far western analysis with purified proteins and immunoprecipitation experiments using purified WRN fragments and Ku subunits expressed in insect cells. Our results are in sharp contrast to a previous study, using in vitro
translated proteins, arguing that WRN associates with Ku only through an interaction between the N-terminus of WRN and a portion of Ku80 (12
). These different results might be due to misfolding of Ku subunits in the in vitro
expression systems that presumably does not occur when Ku is expressed in living cells.
The exact cellular roles of WRN helicase and exonuclease are not yet clear, but recent studies from several laboratories indicate that WRN might play an important role in general DNA metabolism. Regulation of WRN catalytic activities seems to be important in maintaining genomic stability. Several important cellular proteins interact with WRN through its C- or N-terminus and some of them modulate its catalytic activities. For example, p53 interacts directly with the C-terminus of WRN and this interaction inhibits WRN exonuclease activity (8
). The C-terminus of WRN also interacts with human FEN1 and stimulates its cleavage activity on a flap substrate (9
). The N-terminus of WRN physically interacts with PCNA (22
), which forms a ring around duplex DNA. This stabilizes the association of DNA polymerase δ with DNA during replication and increases DNA polymerase δ processivity (23
Our observations here as well as another recent study (11
) demonstrate that the N-terminus of WRN interacts with Ku. Further, we have presented evidence in this report that both the C- and N-termini of WRN interact distinctly with the Ku70 and Ku80 subunits, respectively. Recent characterization of the structure of Ku indicates that this factor similarly forms a ring around DNA helices, much as does PCNA, and suggests that Ku and PCNA might act as interchangeable processivity factors for WRN activity (24
A short central region of Ku70 and Ku80 mediates heterodimerization (25
). The Ku70 heterodimerization domain (Ku70ΔCΔN) paired with full-length Ku80 fails to stimulate WRN exonuclease activity, yet it can interact with WRN though a Ku80–C-WRN interaction. In contrast, a truncation of the C-terminal 178 amino acids of Ku80 paired with full-length Ku70 disrupts the interaction with the C-terminal fragment of WRN, but does not affect interaction with the N-WRN fragment or the ability of Ku to stimulate exonuclease activity. The interaction between Ku80 and WRN is thus not sufficient for stimulation of exonuclease activity. Rather, the inability of the Ku70ΔNΔC/Ku80 heterodimer to stimulate exonuclease activity indicates that either the ability of Ku to bind DNA (missing in Ku70ΔNΔC/Ku80; Fig. A) or the Ku70–N-WRN interaction is functionally critical.
The C-terminal domain of Ku80 is required for interaction with DNA-PKcs (26
) as well as with the C-terminal domain of WRN. Recent work from this laboratory also indicates that DNA-PKcs inactivates WRN exonuclease activity by phosphorylation (27
). We suggest that the interaction between Ku80 and C-WRN may help WRN evade this DNA-PKcs-dependent inactivation, allowing WRN to compete with the ability of DNA-PKcs to interact with Ku.
The physical interaction between N-WRN and Ku70 enhances the nucleolytic processing of DNA ends by WRN. This interaction brings the N-terminus of WRN into close proximity to the DNA. For NHEJ, the excess sequences beyond the region of microhomology should then be trimmed off. Thus, it is tempting to speculate that WRN exonuclease activity may be necessary in processing DNA ends before ligation, and we propose that interaction between WRN and Ku is important to facilitate the nucleolytic processing of ends often observed prior to NHEJ-mediated DSB repair. A recent study showed greater fragmentation of chromosomes in WS cells after ionizing radiation (28
), further strengthening this possibility. Other recent studies support the notion that WRN could be involved in recombination (29
). WS cells are hypersensitive to some kinds of DNA damage that cause DNA interstrand cross-links (30
) and we have recently found that WS cells are deficient in the processing of psoralen-induced DNA interstrand cross-links (31
). Thus, WRN may participate in recombinational repair via both homologous and non-homologous rejoining. The ability of Ku to facilitate WRN exonuclease bypass of certain types of oxidative DNA base lesions could suggest additional flexibility in the processing of ends that might be important for repair of radiation-induced DNA breaks.
While Ku and WRN are likely involved in DNA repair, they may also participate in other processes. For example, a study has shown that Ku participates in transcription (32
) and we have previously reported that WRN protein participates in transcription, both in vitro
and in vivo
). Ku also locates at the telomeric ends (35
), where its interaction with WRN may be biologically important. The WRN homolog sgs1 from Saccharomyces cerevisiae
also locates to telomeric ends (36
Interactions between WRN and several cellular proteins strongly suggest that WRN plays an important role in cellular metabolism, including transcription, replication, repair and/or recombination. A better understanding of WRN function is necessary to address how a loss of WRN causes WS cellular and clinical phenotypes, including increased risk of cancer and age-associated diseases.