Coordinate action of the helicase and 3 ' to 5 ' exonuclease of Werner syndrome protein

Werner syndrome is a human disorder characterized by premature aging, genom ic instability, and abnormal telomere metabolism. The Werner syndrome prote in (WRN) is the only known member of the RecQ DNA helicase family that cont ains a 3' --> 5'-exonuclease. However, it is not known whether both activit ies coordinate in a biological pathway. Here, we describe DNA structures, f orked duplexes containing telomeric repeats, that are substrates for the si multaneous action of both WRN activities. We used these substrates to study the interactions between the WRN helicase and exonuclease on a single DNA molecule. WRN helicase unwinds at the forked end of the substrate, whereas the WRN exonuclease acts at the blunt end. Progression of the WRN exonuclea se is inhibited by the action of WRN helicase converting duplex DNA to sing le strand DNA on forks of various duplex lengths. The WRN helicase and exon uclease act in concert to remove a DNA strand from a long forked duplex tha t is not completely unwound by the helicase. We analyzed the simultaneous a ction of WRN activities on the long forked duplex in the presence of the WR N protein partners, replication protein A (RPA), and the Ku70/80 heterodime r. RPA stimulated the WRN helicase, whereas Ku stimulated the WRN exonuclea se. In the presence of both RPA and Ku, the WRN helicase activity dominated the exonuclease activity.