Moreover, WRN colocalizes and interacts with RAD52 and has strand annealing activity in addition to its DNA unwinding activity (Baynton et al 2003; Machwe et al 2005, 2006). WRN-dependent unwinding activity is signiﬁcantly stronger than previously believed (Machwe et al 2006). Post-translational modiﬁcations In response to DNA damage, WRN can be modulated by post-translational modiﬁcations, including phosphoryla- tion, sumoylation, and acetylation. It is phosphorylated at serine/threonine and tyrosine residues in vivo after bleomycin treatment or after replication stress (Karmakar et al 2002; Cheng et al 2003; Pichierri et al 2003). WRN phosphorylation at serine/threonine residues is primarily dependent on DNA-PKcs and c-Abl kinases (Karmakar et al 2002; Cheng et al 2003; Pichierri et al 2003). Impor- tantly, the serine/threonine phosphorylation of WRN by the DNA-PK complex results in inhibition of both WRN helicase and exonuclease activities, whereas dephosphory- lation of WRN enhances both these activities. Thus, the serine/threonine phosphorylation status of WRN plays a role in the regulation of its catalytic activities (Karmakar et al 2002; Cheng et al 2003). c-Abl was also found to phosphorylate WRN (Cheng et al 2003). c-Abl kinase, a regulator of the DNA damage response, mediates WRN nuclear localization and catalytic activities in response to DNA damage. WRN directly binds to c-Abl and this interaction is disrupted in the early cellular response to bleomycin (Cheng et al 2003). As with phosphorylation by the DNA-PK complex, c-Abl phosphorylation inhibits WRN helicase and exonuclease activities (Cheng et al 2003). Phosphorylation of WRN may inﬂuence other forms of WRN post-translational modiﬁcation, such as sumoylation and/or acetylation. Modiﬁcation of proteins by the small ubiquitin-related modiﬁer 1 (SUMO-1) conjugating system requires a set of enzymes, including SUMO-activating (E1), conjugating (Ubc9), and ligating enzymes (Muller et al 2004). WRN interacts with Ubc9, which is required for conjugation of SUMO-1 to the N-terminal fragment (amino acids 272–514) of WRN (Kawabe et al 2000). In addition, binding of p14 Arf to WRN is multivalent and resembles the binding of p14 Arf to Mdm2, promoting sumoylation of WRN in a synergistic manner with the SUMO-conjugating enzyme Ubc9 (Woods et al 2004). p14 Arf causes redistribution of WRN within the nucleus, and this effect is reversed by expression of a SUMO-speciﬁc Clinical Interventions in Aging 2008:3(3) Werner’s and Hutchinson-Gilford progeria syndromes protease, thus implicating the SUMO conjugation pathway in controlling WRN re-localization (Woods et al 2004). Besides phosphorylation and sumoylation, WRN can be acetylated after mitomycin C or methyl methane-sulfonate treatment (Sharma et al 2005). On exposure to UV or ionizing radiation, WRN is acetylated by acetyltransferase p300, a transcriptional coactivator, which has acetylation activity (Blander et al 2002). DNA replication WRN helicase unwinds replication fork structures very efﬁciently (Mohaghegh et al 2001), acting to resolve the block and/or in the replication restart process (Rodríguez- López et al 2003). Compared to normal cells, cells from WS patients undergo premature replicative senescence (Martin et al 1970; Salk et al 1985), display an extended S-phase (Poot et al 1992), and show a reduced frequency of replication initiation sites (Takeuchi et al 1982; Hanaoka et al 1985), thus exhibiting defects in DNA replication consistent with the inability to properly recover from DNA replication fork demise (Hickson 2003). WRN is able to interact with PCNA, DNA topoisomer- ase I (topo I), polδ, and RPA (Wold 1997; Lebel and Leder 1998; Brosh et al 1999; Lebel et al 1999; Constantinou et al 2000; Huang et al 2000; Laine et al 2003) and catalyze DNA unwinding in vitro for DNA replication, recombination, and repair (Brosh and Bohr 2002). WRN and PCNA colocalize at replication foci, suggesting a physiological interaction between them in cycling primary cells (Rodríguez-López et al 2003). In addition, WRN is as part of the 17S multiprotein DNA replication complex, and establishes PCNA and topoisomerase I as the two WRN- interacting components (Warbrick 1998, 2000; Lebel et al 1999; Rodríguez-López et al 2003). Polδ participates in DNA replication and repair of DNA damage. WRN interacts speciﬁcally with the p50 subunit of polδ, and WRN directly modiﬁes DNA replication via its interaction with p50 and is involved in the dynamic relocal- ization of polδ complexes within the nucleus (Kamath-Loeb et al 2000; Szekely et al 2000). Moreover, WRN enhances the rate of nucleotide incorporation in polδ-mediated replication in the absence of PCNA and its helicase activity enables polδ to overcome hairpin and G-quadruplex DNA structures (Fry and Loeb 1999; Szekely et al 2000; Kamath-Loeb et al 2000, 2001; Brosh et al 2001b; Mohaghegh et al 2001). Thus, WRN may facilitate polδ-mediated DNA replication, and disruption of the WRN-polδ interaction in WS cells may contribute to the S-phase defects. 433