TRF1 and TRF2 binding to telomeres is modulated by nucleosomal organization

The ends of eukaryotic chromosomes need to be protected from the activation of a DNA damage response that leads the cell to replicative senescence or apoptosis. In mammals, protection is accomplished by a six-factor complex named shelterin, which organizes the terminal TTAGGG repeats in a still ill-defined structure, the telomere. The stable interaction of shelterin with telomeres mainly depends on the binding of two of its components, TRF1 and TRF2, to double-stranded telomeric repeats. Tethering of TRF proteins to telomeres occurs in a chromatin environment characterized by a very compact nucleosomal organization. In this work we show that binding of TRF1 and TRF2 to telomeric sequences is modulated by the histone octamer. By means of in vitro models, we found that TRF2 binding is strongly hampered by the presence of telomeric nucleosomes, whereas TRF1 binds efficiently to telomeric DNA in a nucleosomal context and is able to remodel telomeric nucleosomal arrays. Our results indicate that the different behavior of TRF proteins partly depends on the interaction with histone tails of their divergent N-terminal domains. We propose that the interplay between the histone octamer and TRF proteins plays a role in the steps leading to telomere deprotection

TRF2 controls telomeric nucleosome organization in a cell cycle phase-dependent manner

Mammalian telomeres stabilize chromosome ends as a result of their assembly into a peculiar form of chromatin comprising a complex of non-histone proteins named shelterin. TRF2, one of the shelterin components, binds to the duplex part of telomeric DNA and is essential to fold the telomeric chromatin into a protective cap. Although most of the human telomeric DNA is organized into tightly spaced nucleosomes, their role in telomere protection and how they interplay with telomere-specific factors in telomere organization is still unclear. In this study we investigated whether TRF2 can regulate nucleosome assembly at telomeres.By means of chromatin immunoprecipitation (ChIP) and Micrococcal Nuclease (MNase) mapping assay, we found that the density of telomeric nucleosomes in human cells was inversely proportional to the dosage of TRF2 at telomeres. This effect was not observed in the G1 phase of the cell cycle but appeared coincident of late or post-replicative events. Moreover, we showed that TRF2 overexpression altered nucleosome spacing at telomeres increasing internucleosomal distance. By means of an in vitro nucleosome assembly system containing purified histones and remodeling factors, we reproduced the short nucleosome spacing found in telomeric chromatin. Importantly, when in vitro assembly was performed in the presence of purified TRF2, nucleosome spacing on a telomeric DNA template increased, in agreement with in vivo MNase mapping.Our results demonstrate that TRF2 negatively regulates the number of nucleosomes at human telomeres by a cell cycle-dependent mechanism that alters internucleosomal distance. These findings raise the intriguing possibility that telomere protection is mediated, at least in part, by the TRF2-dependent regulation of nucleosome organization

GEN1 from a thermophilic fungus is functionally closely similar to non-eukaryotic junction-resolving enzymes

AbstractProcessing of Holliday junctions is essential in recombination. We have identified the gene for the junction-resolving enzyme GEN1 from the thermophilic fungus Chaetomium thermophilum and expressed the N-terminal 487-amino-acid section. The protein is a nuclease that is highly selective for four-way DNA junctions, cleaving 1nt 3′ to the point of strand exchange on two strands symmetrically disposed about a diagonal axis. CtGEN1 binds to DNA junctions as a discrete homodimer with nanomolar affinity. Analysis of the kinetics of cruciform cleavage shows that cleavage of the second strand occurs an order of magnitude faster than the first cleavage so as to generate a productive resolution event. All these properties are closely similar to those described for bacterial, phage and mitochondrial junction-resolving enzymes. CtGEN1 is also similar in properties to the human enzyme but lacks the problems with aggregation that currently prevent detailed analysis of the latter protein. CtGEN1 is thus an excellent enzyme with which to engage in biophysical and structural analysis of eukaryotic GEN1

NMR studies of telomeric nucleoprotein complexes involving the Myb-like domain of the human telomeric protein TRF2

In order to study the binding of the Myb-like domain of the human telomeric protein TRF2 (Myb-TRF2) with different structural components of the t-loop model, we report NMR studies of the binding of Myb-TRF2 protein with two repeats human telomeric DNA under three conformations. Our results showed that Myb-TRF2 binds to the duplex and even to the quadruplex and the random coil G-rich strand. The solution structure of Myb-TRF2 reported here looks like Myb-TRF1 suggesting similar DNA binding mode. As a matter of fact, we have shown that its binding to the human telomeric duplex presents great similarities with this of Myb-TRF1

A higher-order entity formed by the flexible assembly of RAP1 with TRF2

Essonne committee of the Ligue contre le cancer M18756 M22897 Foundation ARC pour la Recherche sur le Cancer SFI20121205503International audienceTelomere integrity is essential to maintain genome stability, and telomeric dysfunctions are associated with cancer and aging pathologies. In human, the shelterin complex binds TTAGGG DNA repeats and provides capping to chromosome ends. Within shel-terin, RAP1 is recruited through its interaction with TRF2, and TRF2 is required for telomere protection through a network of nucleic acid and protein interactions. RAP1 is one of the most conserved shelterin proteins although one unresolved question is how its interaction may influence TRF2 properties and regulate its capacity to bind multiple proteins. Through a combination of biochemical, biophysical and structural approaches, we unveiled a unique mode of assembly between RAP1 and TRF2. The complete interaction scheme between the full-length proteins involves a complex biphasic interaction of RAP1 that directly affects the binding properties of the assembly. These results reveal how a non-DNA binding protein can influence the properties of a DNA-binding partner by mutual conformational adjustments

Crystal Structure of a Eukaryotic GEN1 Resolving Enzyme Bound to DNA

SummaryWe present the crystal structure of the junction-resolving enzyme GEN1 bound to DNA at 2.5 Å resolution. The structure of the GEN1 protein reveals it to have an elaborated FEN-XPG family fold that is modified for its role in four-way junction resolution. The functional unit in the crystal is a monomer of active GEN1 bound to the product of resolution cleavage, with an extensive DNA binding interface for both helical arms. Within the crystal lattice, a GEN1 dimer interface juxtaposes two products, whereby they can be reconnected into a four-way junction, the structure of which agrees with that determined in solution. The reconnection requires some opening of the DNA structure at the center, in agreement with permanganate probing and 2-aminopurine fluorescence. The structure shows that a relaxation of the DNA structure accompanies cleavage, suggesting how second-strand cleavage is accelerated to ensure productive resolution of the junction

Walking of antitumor bifunctional trinuclear PtII complex on double-helical DNA

The trinuclear BBR3464 ([{trans-PtCl(NH3)2}2µ-(trans-Pt(NH3)2(H2N(CH2)6NH2)2)]4+) belongs to the polynuclear class of platinum-based anticancer agents. DNA adducts of this complex differ significantly in structure and type from those of clinically used mononuclear platinum complexes, especially, long-range (Pt, Pt) intrastrand and interstrand cross-links are formed in both 5′–5′ and 3′–3′ orientations. We show employing short oligonucleotide duplexes containing single, site-specific cross-links of BBR3464 and gel electrophoresis that in contrast to major DNA adducts of clinically used platinum complexes, under physiological conditions the coordination bonds between platinum and N7 of G residues involved in the cross-links of BBR3464 can be cleaved. This cleavage may lead to the linkage isomerization reactions between this metallodrug and double-helical DNA. Differential scanning calorimetry of duplexes containing single, site-specific cross-links of BBR3464 reveals that one of the driving forces that leads to the lability of DNA cross-links of this metallodrug is a difference between the thermodynamic destabilization induced by the cross-link and by the adduct into which it could isomerize. The rearrangements may proceed in the way that cross-links originally formed in one strand of DNA can spontaneously translocate from one DNA strand to its complementary counterpart, which may evoke walking of the platinum complex on DNA molecule

The N-terminal domains of TRF1 and TRF2 regulate their ability to condense telomeric DNA

TRF1 and TRF2 are key proteins in human telomeres, which, despite their similarities, have different behaviors upon DNA binding. Previous work has shown that unlike TRF1, TRF2 condenses telomeric, thus creating consequential negative torsion on the adjacent DNA, a property that is thought to lead to the stimulation of single-strand invasion and was proposed to favor telomeric DNA looping. In this report, we show that these activities, originating from the central TRFH domain of TRF2, are also displayed by the TRFH domain of TRF1 but are repressed in the full-length protein by the presence of an acidic domain at the N-terminus. Strikingly, a similar repression is observed on TRF2 through the binding of a TERRA-like RNA molecule to the N-terminus of TRF2. Phylogenetic and biochemical studies suggest that the N-terminal domains of TRF proteins originate from a gradual extension of the coding sequences of a duplicated ancestral gene with a consequential progressive alteration of the biochemical properties of these proteins. Overall, these data suggest that the N-termini of TRF1 and TRF2 have evolved to finely regulate their ability to condense DNA

Rif1 Supports the Function of the CST Complex in Yeast Telomere Capping

Telomere integrity in budding yeast depends on the CST (Cdc13-Stn1-Ten1) and shelterin-like (Rap1-Rif1-Rif2) complexes, which are thought to act independently from each other. Here we show that a specific functional interaction indeed exists among components of the two complexes. In particular, unlike RIF2 deletion, the lack of Rif1 is lethal for stn1ΔC cells and causes a dramatic reduction in viability of cdc13-1 and cdc13-5 mutants. This synthetic interaction between Rif1 and the CST complex occurs independently of rif1Δ-induced alterations in telomere length. Both cdc13-1 rif1Δ and cdc13-5 rif1Δ cells display very high amounts of telomeric single-stranded DNA and DNA damage checkpoint activation, indicating that severe defects in telomere integrity cause their loss of viability. In agreement with this hypothesis, both DNA damage checkpoint activation and lethality in cdc13 rif1Δ cells are partially counteracted by the lack of the Exo1 nuclease, which is involved in telomeric single-stranded DNA generation. The functional interaction between Rif1 and the CST complex is specific, because RIF1 deletion does not enhance checkpoint activation in case of CST-independent telomere capping deficiencies, such as those caused by the absence of Yku or telomerase. Thus, these data highlight a novel role for Rif1 in assisting the essential telomere protection function of the CST complex