Trapping Rap1 at the telomere to prevent chromosome end fusions.

The prevention of nonhomologous end-joining (NHEJ) reactions between chromosome ends is crucial for maintaining genome stability. Although this protective function is known to be fulfilled by a core of conserved telomeric proteins that are collectively known as Shelterin, its mechanistic details remain a mystery. In this issue, Sarthy et al (2009) lend fresh insight by developing an ingenious method to dissect the role of hRAP1 in preventing telomeric NHEJ independently of other Shelterin components. Nearly 70 years ago, McClintock (1941) and Muller (1938) deduced that natural chromosome ends are distinct from the ends created by chromosome breakage in their ability to avoid fusion reactions. The lethal consequences of telomere dysfunction are graphically illustrated by more recent studies in which loss of the human telomere repeat-binding protein TRF2 leads to ‘trains’ of chromosomes hitched together through NHEJ (Celli and de Lange, 2005). Although these studies show the central role of TRF2 in the defining protective function of telomeres, the multitude of molecular interactions in which TRF2 engages has confounded an understanding of precisely how it prevents NHEJ. Rap1 has been both one of the most well-studied and enlightening of telomeric components and one of the most enigmatic. Budding yeast Rap1 (ScRap1) binds directly to telomeric repeats (as well as numerous promoters) and coordinates several telomeric functions, including silencing and telomerase regulation. Moreover, landmark studies have shown that ScRap1 prevents NHEJ between chromosome ends (Pardo and Marcand, 2005). Interestingly, although both human and fission yeast Rap1 were identified by their homology to ScRap1, they lack DNA-binding ability and associate with telomeres through interactions with the related telomere-binding proteins TRF2 and Taz1, respectively. Indeed, loss of either Taz1 or Rap1 in fission yeast leads to NHEJ-mediated telomere fusions (Ferreira and Cooper, 2001; Miller et al, 2005). Technical challenges to developing a mammalian RAP1 knockout model have hindered our ability to extend this idea to metazoans (Tan et al, 2003). However, by combining the insights gained from previous in vitro experiments with an innovative approach to constructing informative chimeric molecules, Sarthy et al have pinpointed a role of hRAP1 in NHEJ inhibition. The Baumann lab previously addressed the minimal elements required for protection from chromosomal endjoining in human cell extracts (Bae and Baumann, 2007). They found that as few as eight telomeric (TTAGGG) repeats were sufficient to block the nearby NHEJ. As this tract length is too short to confer t-loops, these observations suggested a direct NHEJ-blocking function that would not rely on telomeric higher-order structure. Immunodepletion and add-back of specific proteins showed that protection depends on both TRF2 and hRAP1. However, as hRAP1 fails to localize to telomeres in the absence of TRF2, the sufficiency of hRAP1 for blocking NHEJ could not be addressed. To circumvent this problem, Sarthy et al devised a way to target hRAP1 to