DNA structure-dependent recruitment of telomeric proteins to single-stranded/double-stranded DNA junctions (original) (raw)
Related papers
The effect of the TRF2 N-terminal and TRFH regions on telomeric G-quadruplex structures
Nucleic Acids Research, 2009
The sequence of human telomeric DNA consists of tandem repeats of 5'-d(TTAGGG)-3'. This guaninerich DNA can form G-quadruplex secondary structures which may affect telomere maintenance. A current model for telomere protection by the telomere-binding protein, TRF2, involves the formation of a t-loop which is stabilized by a strand invasion-like reaction. This type of reaction may be affected by G-quadruplex structures. We analyzed the influence of the arginine-rich, TRF2 N-terminus (TRF2 B ), as well as this region plus the TRFH domain of TRF2 (TRF2 BH ), on the structure of G-quadruplexes. Circular dichroism results suggest that oligonucleotides with 4, 7 and 8 5'-d(TTAGGG)-3' repeats form hybrid structures, a mix of parallel/ antiparallel strand orientation, in K + . TRF2 B stimulated the formation of parallel-stranded structures and, in some cases, intermolecular structures. TRF2 BH also stimulated intermolecular but not parallel-stranded structures. Only full-length TRF2 and TRF2 BH stimulated uptake of a telomeric single-stranded oligonucleotide into a plasmid containing telomeric DNA in the presence of K + . The results in this study suggest that G-quadruplex formation inhibits oligonucleotide uptake into the plasmid, but the inhibition can be overcome by TRF2. This study is the first analysis of the effects of TRF2 domains on G-quadruplex structures and has implications for the role of G-quadruplexes and TRF2 in the formation of t-loops.
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.
Telomere end-binding proteins control the formation of G-quadruplex DNA structures in vivo
Nature Structural & Molecular Biology, 2005
Telomere end-binding proteins (TEBPs) bind to the guanine-rich overhang (G-overhang) of telomeres. Although the DNA binding properties of TEBPs have been investigated in vitro, little is known about their functions in vivo. Here we use RNA interference to explore in vivo functions of two ciliate TEBPs, TEBPa and TEBPb. Silencing the expression of genes encoding both TEBPs shows that they cooperate to control the formation of an antiparallel guanine quadruplex (G-quadruplex) DNA structure at telomeres in vivo. This function seems to depend on the role of TEBPa in attaching telomeres in the nucleus and in recruiting TEBPb to these sites. In vitro DNA binding and footprinting studies confirm the in vivo observations and highlight the role of the C terminus of TEBPb in G-quadruplex formation. We have also found that G-quadruplex formation in vivo is regulated by the cell cycle-dependent phosphorylation of TEBPb.
Angewandte Chemie International Edition, 2009
Telomere structures are essential for genome stability. [1] Telomere dysfunction can cause cell senescence, death, or genomic instability. [1] Human telomeric DNA consists of a duplex region composed of TTAGGG repeats, ending in a 100-200 nucleotide (nt) G-rich single-stranded overhang. [1] The telomeric overhang is recognized as a critical component of telomere structures, required for telomere end protection and extension. [1] It is also particularly attractive as a target for anticancer therapeutic development. [2] The detailed structural features and mechanisms of telomeric overhang that are responsible for protecting the chromosome ends are still insufficiently understood, although several alternative structures have been proposed for human telomeric DNA, such as G-quadruplex or T-loop. [3, 4] The T-loop and G-quadruplex structural models with the same human telomere sequence seem to leave many questions unanswered. Some important experimental findings are not adequately explained by these models. Among those findings are the following: 1) The T-loop structure, which is stabilized by the 3'-overhang strand encroaching into the double-stranded region, cannot explain the inhibition of telomerase activity by a G-quadruplex stabilizing molecule. [2] 2) Previous studies showed that only one TTAGGG repeat (6 nt) of 3'-overhang is sufficient for T-loop formation. [5] However, a recent study showed that, in most senescent cells, telomeric overhangs are still long, often averaging 50-100 nt. [6] This finding raises the question of why that DNA length, seemingly sufficient for T-loop formation, does not provide a protective structure for prevention of senescence in cells. For the G-quadruplex structure, most previous studies focused on individual G-quadruplexes formed by short telomeric DNA (especially 22 nt unimolecular G-quadruplexes). [3] However, it might be more biologically relevant to take into account the real length of the 3'-terminal singlestranded overhang of human telomeric DNA in vivo (100-200 nt) to directly reveal the structural features of long
TRF2/RAP1 and DNA–PK mediate a double protection against joining at telomeric ends
The EMBO Journal, 2010
DNA-dependent protein kinase (DNA-PK) is a double-strand breaks repair complex, the subunits of which (KU and DNA-PKcs) are paradoxically present at mammalian telomeres. Telomere fusion has been reported in cells lacking these proteins, raising two questions: how is DNA-PK prevented from initiating classical ligase IV (LIG4)-dependent non-homologous end-joining (C-NHEJ) at telomeres and how is the backup end-joining (EJ) activity (B-NHEJ) that operates at telomeres under conditions of C-NHEJ deficiency controlled? To address these questions, we have investigated EJ using plasmid substrates bearing double-stranded telomeric tracks and human cell extracts with variable C-NHEJ or B-NHEJ activity. We found that (1) TRF2/RAP1 prevents C-NHEJ-mediated end fusion at the initial DNA-PK end binding and activation step and (2) DNA-PK counteracts a potent LIG4-independent EJ mechanism. Thus, telomeres are protected against EJ by a lock with two bolts. These results account for observations with mammalian models and underline the importance of alternative non-classical EJ pathways for telomere fusions in cells. The EMBO Journal VOL 29 | NO 9 | 2010 EMBO THE EMBO JOURNAL THE EMBO JOURNAL
G-quadruplex formation in telomeres enhances POT1/TPP1 protection against RPA binding
Proceedings of the National Academy of Sciences, 2014
Human telomeres terminate with a single-stranded 3′ G overhang, which can be recognized as a DNA damage site by replication protein A (RPA). The protection of telomeres (POT1)/POT1-interacting protein 1 (TPP1) heterodimer binds specifically to single-stranded telomeric DNA (ssTEL) and protects G overhangs against RPA binding. The G overhang spontaneously folds into various G-quadruplex (GQ) conformations. It remains unclear whether GQ formation affects the ability of POT1/TPP1 to compete against RPA to access ssTEL. Using single-molecule Förster resonance energy transfer, we showed that POT1 stably loads to a minimal DNA sequence adjacent to a folded GQ. At 150 mM K + , POT1 loading unfolds the antiparallel GQ, as the parallel conformation remains folded. POT1/TPP1 loading blocks RPA's access to both folded and unfolded telomeres by two orders of magnitude. This protection is not observed at 150 mM Na + , in which ssTEL forms only a lessstable antiparallel GQ. These results suggest that GQ formation of telomeric overhangs may contribute to suppression of DNA damage signals.
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