TRF1 promotes parallel pairing of telomeric tracts in vitro (original) (raw)
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TRF1 is a dimer and bends telomeric DNA
The EMBO Journal, 1997
polarity towards the end of the chromosome (Moyzis USA et al., 1988;. This sequence is 1 Corresponding author maintained by telomerase, a ribonucleoprotein that uses an internal RNA template to synthesize tandem arrays of TRF1 is a mammalian telomeric protein that binds to telomeric repeats onto chromosome ends ; the duplex array of TTAGGG repeats at chromosome . The TTAGGG repeat arrays are the ends. TRF1 has homology to the DNA-binding domain only DNA requirement for telomere function in somatic of the Myb family of transcription factors but, unlike human cells ; Barnett most Myb-related proteins, TRF1 carries one rather . than multiple Myb-type DNA-binding motifs. Here we
Interactions of TRF2 with model telomeric ends
Biochemical and Biophysical Research Communications, 2007
Telomeres are DNA-protein complexes at the ends of eukaryotic chromosomes, the integrity of which is essential for chromosome stability. An important telomere binding protein, TTAGGG repeat factor 2 (TRF2), is thought to protect telomere ends by remodeling them into T-loops. We show that TRF2 specifically interacts with telomeric ss/ds DNA junctions and binding is sensitive to the sequence of the 3 0 , guanine-strand (G-strand) overhang and double-stranded DNA sequence at the junction. Association of TRF2 with DNA junctions hinders cleavage by exonuclease T. TRF2 interactions with the G-strand overhang do not involve the TRF2 DNA binding domain or the linker region. However, mobility shifts and atomic force microscopy show that the previously uncharacterized linker region is involved in DNA-specific, TRF2 oligomerization. We suggest that T-loop formation at telomere ends involves TRF2 binding to the G-strand overhang and oligomerization through both the known TRFH domain and the linker region.
Structure of the TRFH Dimerization Domain of the Human Telomeric Proteins TRF1 and TRF2
Molecular Cell, 2001
apoptosis (Karlseder et al., 1999; van Steensel et al., Hills Road 1998). In general, mammalian cells respond to TRF2 Cambridge CB2 2QH deficiency as if their natural chromosome ends resemble United Kingdom DNA breaks, resulting in degradation of the single-2 The Rockefeller University stranded telomeric overhang, inappropriate ligation of New York, New York 10021 chromosome ends, and activation of the ATM/p53 DNA damage response pathway (Karlseder et al., 1999; van Steensel et al., 1998). Summary Similarly, fission yeast telomeres are protected from end-to-end fusions and recombination by a TRF-related TRF1 and TRF2 are key components of vertebrate teloprotein, Taz1, that binds to duplex telomeric DNA (Fermeres. They bind to double-stranded telomeric DNA reira and Cooper, 2001; Nakamura et al., 1998). Like as homodimers. Dimerization involves the TRF homol-TRF1 and TRF2, Taz1 acts as a negative regulator of ogy (TRFH) domain, which also mediates interactions telomere length (Cooper et al., 1997; Nimmo et al., 1998). with other telomeric proteins. The crystal structures Remarkably, budding yeast lacks a TRF-like telomeric of the dimerization domains from human TRF1 and protein, and in this organism scRap1p is the main factor TRF2 were determined at 2.9 and 2.2 Å resolution, that binds duplex telomeric DNA and regulates telomere respectively. Despite a modest sequence identity, the length (Marcand et al., 1997a; 1997b; McEachern et al., two TRFH domains have the same entirely ␣-helical 2000). architecture, resembling a twisted horseshoe. The di-The TRF family of proteins have similar architectures, merization interfaces feature unique interactions that defined by two sequence features (Figure 1A). First, prevent heterodimerization. Mutational analysis of these proteins have C-terminal DNA binding motifs that TRF1 corroborates the structural data and underare closely related to the Myb domain of c-Myb (Nishiscores the importance of the TRFH domain in dimerkawa et al., 1998; Ogata et al., 1994) and also to the ization, DNA binding, and telomere localization. A pos-Myb/homeodomains of the budding yeast Rap1p (Konig sible structural homology between the TRFH domain et al., 1996; Konig and Rhodes, 1997). Second, these of fission yeast telomeric protein Taz1 with those of proteins have a centrally located sequence motif of the vertebrate TRFs is suggested. about 200 aa, referred to as the TRF homology (TRFH) domain that is unique to this gene family (Broccoli et
TRF1 and TRF2 binding to telomeres is modulated by nucleosomal organization
Nucleic Acids Research, 2015
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.
A human interstitial telomere associates in vivo with specific TRF2 and TIN2 proteins
European Journal of Human Genetics, 2002
Mammalian telomeres are composed of long arrays of TTAGGG repeats that form a nucleoprotein complex which protects the chromosome ends. Human telomere function is known to require two TTAGGG repeat factors, TRF1 and TRF2, and several interacting proteins, but the mechanism by which the DNA/protein complex prevents end to end fusion in vivo has not been elucidated. In order to better understand the role of specific telomere-associated proteins in the organisation of chromosome ends, we have studied a patient with a rare chromosome rearrangement that has given rise to an interstitial telomere. Using specific antibodies and immuno-FISH on unfixed metaphase chromosomes, we show that the proteins TRF2 and TIN2 (TIN2 interacts with TRF1) co-localise with the interstitial TTAGGG repeats. Our results demonstrate, for the first time in humans, that TRF2 and TIN2 proteins associate with interstitial duplex TTAGGG repeats, in vivo. They confirm that double stranded-telomeric repeats, even when complexed with specific proteins, are not sufficient to create a functional telomere. Finally, they suggest a possible role for proteins in stabilising interstitial TTAGGG repeats.
Human Molecular Genetics, 1997
Mammalian chromosome ends contain long arrays of TTAGGG repeats that are complexed to a telomere specific protein, the TTAGGG repeat binding factor, TRF1. Here we describe the characterization of genes encoding the human and mouse TRF1 proteins, hTRF1 and mTRF1. The mTRF1 cDNA was isolated based on sequence similarity to the hTRF1 cDNA and the mTRF1 mRNA was shown to be ubiquitously expressed as a single 1.9 kb polyadenylated transcript in mouse somatic tissues. High levels of a 2.1 kb transcript were found in testes. In vitro translation of the mTRF1 cDNA resulted in a 56 kDa protein that binds to TTAGGG repeat arrays. mTRF1 displayed the same sequence specificity as hTRF1, preferring arrays of TTAGGG repeats as a binding substrate over TTAGGC and TTGGGG repeats. Expression of an epitope-tagged version of mTRF1 showed that the protein is located at the ends of murine metaphase chromosomes. In agreement, conceptual translation indicated that mTRF1 and hTRF1 are similarly-sized proteins with nearly identical C-terminal Myb-related DNA binding motifs. In addition, comparison of the predicted mTRF1 and hTRF1 amino acid sequences showed that the acidic nature of the N-terminus of TRF1 is conserved and revealed a highly conserved novel domain of ∼200 amino acids in the middle of the proteins. However, other regions of the proteins are poorly conserved (<35% identity) and the overall level of identity of the mTRF1 and hTRF1 amino acid sequences is only 67%. The TRF1 genes are not syntenic; the hTRF1 gene localized to human chromosome 8 band q13 while the mTRF1 gene localized to mouse chromosome 17 band E3. The data indicate that the genes for mammalian telomeric proteins evolve rapidly.
The Myb/SANT domain of the telomere-binding protein TRF2 alters chromatin structure
Nucleic Acids Research, 2009
Eukaryotic DNA is packaged into chromatin, which regulates genome activities such as telomere maintenance. This study focuses on the interactions of a myb/SANT DNA-binding domain from the telomere-binding protein, TRF2, with reconstituted telomeric nucleosomal array fibers. Biophysical characteristics of the factor-bound nucleosomal arrays were determined by analytical agarose gel electrophoresis (AAGE) and single molecules were visualized by atomic force microscopy (AFM). The TRF2 DNA-binding domain (TRF2 DBD) neutralized more negative charge on the surface of nucleosomal arrays than histone-free DNA. Binding of TRF2 DBD at lower concentrations increased the radius and conformational flexibility, suggesting a distortion of the fiber structure. Additional loading of TRF2 DBD onto the nucleosomal arrays reduced the flexibility and strongly blocked access of micrococcal nuclease as contour lengths shortened, consistent with formation of a unique, more compact higher-order structure. Mirroring the structural results, TRF2 DBD stimulated a strand invasionlike reaction, associated with telomeric t-loops, at lower concentrations while inhibiting the reaction at higher concentrations. Full-length TRF2 was even more effective at stimulating this reaction. The TRF2 DBD had less effect on histone-free DNA structure and did not stimulate the t-loop reaction with this substrate, highlighting the influence of chromatin structure on the activities of DNA-binding proteins.