The nonhomologous end-joining (NHEJ) repair pathway is inhibited at telomeres, preventing chromosome fusion. In budding yeast Saccharomyces cerevisiae, the Rap1 protein directly binds the telomere sequences and is required for NHEJ inhibition. Here we show that the Rap1 C-terminal domain establishes two parallel inhibitory pathways through the proteins Rif2 and Sir4. In addition, the central domain of Rap1 inhibits NHEJ independently of Rif2 and Sir4. Thus, Rap1 establishes several independent pathways to prevent telomere fusions. We discuss a possible mechanism that would explain Rif2 multifunctionality at telomeres and the recent evolutionary origin of Rif2 from an origin recognition complex (ORC) subunit.Supplemental material is available at http://www.genesdev.org.Received August 30, 2007; revised version accepted February 29, 2008. Nonhomologous end-joining (NHEJ) inhibition at telomeres prevents fusions between two telomeres and between a telomere and a double-stranded broken end (DuBois et al. 2002;Chan and Blackburn 2003;Mieczkowski et al. 2003;Riha et al. 2006). In fission yeast Schizosaccharomyces pombe, the Taz1 protein binds telomeric DNA and recruits the Rap1 protein. Both Taz1 and Rap1 are required for NHEJ inhibition (Ferreira and Cooper 2001;Miller et al. 2005). In mammals, TRF1 and TRF2 are the two orthologs of Taz1, and RAP1 is recruited to the telomeres by TRF2 (Li et al. 2000). In vivo, TRF2 is essential for NHEJ inhibition (van Steensel et al. 1998;Celli and de Lange 2005;Celli et al. 2006). In vitro, it has been found that NHEJ inhibition by telomeric repeats requires TRF2 and RAP1 (Bae and Baumann 2007). In Saccharomyces cerevisiae, there is no ortholog of Taz1/ TRF1/TRF2. Rap1 directly binds the telomere sequences, and its loss causes telomere fusions dependent on all the factors known to be required for NHEJ (Pardo and Marcand 2005;Pardo et al. 2006).Rap1 is a modular protein with three conserved domains: a BRCT domain of unknown function in the Nterminal region, a centrally located DNA-binding domain with two Myb-like folds, and an independent Cterminal domain called RCT (Rap1 C-terminal) ( Fig. 1A; Li et al. 2000;Dreesen et al. 2007). In budding yeast, Rap1 also binds to ∼5% of the promoters and is required for viability. The BRCT and RCT domains are dispensable (Kyrion et al. 1992;Moretti et al. 1994). The RCT is required for the negative feedback loop that represses telomere elongation by telomerase (Kyrion et al. 1992;Marcand et al. 1997) and for the establishment of a silent chromatin near telomeres (Kyrion et al. 1993;Buck and Shore 1995). It interacts with the proteins Rif1, Rif2, Sir3, and Sir4. Rif1 and Rif2 are required for the inhibition of telomere elongation (Wotton and Shore 1997; Levy and Blackburn 2004;Teixeira et al. 2004). Sir3 and Sir4 establish transcriptional silencing, which also requires Sir2, a conserved histone deacetylase. In this work, we ask which Rap1 domains and interacting factors are involved in NHEJ inhibition at telomeres. Results and Discussion Rap...

In ciliates, the development of the somatic macronucleus involves the programmed excision of thousands of internal eliminated sequences (IES) scattered throughout the germ line genome. Previous work with Tetrahymena thermophila has suggested that excision is initiated by a staggered double-strand break (DSB) at one IES end. Nucleophilic attack of the other end by the 3OH group carried by the firstly broken chromosome end leads to macronuclear junction closure. In this study, we mapped the 3OH and 5PO 4 groups that are developmentally released at Paramecium IES boundaries, which are marked by two conserved TA dinucleotides, one of which remains in the macronuclear genome after excision. We show that initiating DSBs at both ends generate 4-base 5 overhangs centered on the TA. Based on the observed processing of the 5-terminal residue of each overhang, we present a new model for the precise closure of macronuclear chromosomes in Paramecium tetraurelia, different from that previously proposed for Tetrahymena. In our model, macronucleus-destined broken ends are aligned through the partial pairing of their 5-nTAn-3 extensions and joined after trimming of the 5 flaps.Recombination processes involving the cutting and rejoining of DNA molecules have been found in a wide range of organisms, and two types of molecular mechanisms have been documented (reviewed in reference 5): conservative site-specific recombination, leading to the deletion/insertion or inversion of DNA sequences, involves the transient formation of covalent protein-DNA intermediates, while other reactions, such as the transposition of most class II transposons, generate DNA intermediates. Ciliates provide attractive models for the study of programmed DNA rearrangements and their regulation, because their entire genome is remodeled at each sexual cycle (reviewed in references 13 and 34

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