DNA-RNA hybrid G-quadruplex tends to form near the 3′ end of telomere overhang

Telomeric repeat-containing RNA (TERRA) has been suggested to participate in telomere maintenance. TERRA consisting of UUAGGG repeats is capable of forming an intermolecular G-quadruplex (GQ) with single-stranded TTAGGG-repeat DNA in the telomere 3′ overhang. To explore the structural features and potential functions of this DNA-RNA hybrid GQ (HGQ), we used single-molecule FRET to study the folding patterns of DNA with four to seven telomeric tandem repeats annealed with a short RNA consisting of two or five telomeric repeats. Our data highlight that RNA prefers to form DNA-RNA HGQ near the 3′ end of telomeric DNA. Furthermore, the unfolding of secondary structures by a complementary C-rich sequence was observed for DNA GQ but not for DNA-RNA HGQ, which demonstrated the enhanced stability of the telomere 3′ end via hybridization with RNA. These conformational and physical properties of telomeric DNA-RNA HGQ suggest that TERRA might limit access to the 3′ end of the telomeric DNA overhang, which is known to be critical for the interaction with telomerase and other telomere-associated proteins.     

Generation of telomeric G strand overhangs involves both G and C strand cleavage

Processing of telomeric DNA is required to generate the 3' G strand overhangs necessary for capping chromosome ends. We have investigated the steps involved in telomere processing by examining G overhang structure in Tetrahymena cells that lack telomerase or have altered telomeric sequences. We show that overhangs are generated by two precise cleavage steps involving nucleases that are robust but lack sequence specificity. Our data suggest that a G overhang binding protein delineates the boundaries for G and C strand cleavage. We also show that telomerase is not the nuclease responsible for G strand cleavage, although telomerase depletion alters the precision of processing. This change in processing indicates that telomerase affects multiple transactions at the telomere and provides a physical footprint for the continued association of telomerase with the telomere after repeat addition is complete.     

Utilization of ribonucleotides and RNA primers by Tetrahymena telomerase.

Telomerase is a ribonucleoprotein (RNP) DNA polymerase involved in telomere synthesis. A short sequence within the telomerase RNA component provides a template for de novo addition of the G-rich strand of a telomeric simple sequence repeat onto chromosome termini. In vitro, telomerase can elongate single-stranded DNA primers processively: one primer can be extended by multiple rounds of template copying before product dissociation. Telomerase will incorporate dNTPs or ddNTPs and will elongate any G-rich, single-stranded primer DNA. In this report, we show that Tetrahymena telomerase was able to incorporate a ribonucleotide, rGTP, into product polynucleotide. Synthesis of the product [d(TT)r(GGGG)]n was processive, suggesting that the chimeric product remained associated with the enzyme both at the active site and at a second, previously characterized, template-independent product binding site. As predicted by this finding, RNA-containing oligonucleotides served as primers for elongation. More than 3 nt of RNA at a primer 3' end decreased the quantity of product synthesis but increased the affinity of the primer for telomerase. Thus, RNA-containing primers were effective as competitive inhibitors of DNA primer elongation by telomerase. These results support the possible evolutionary origin of telomerase as an RNA-dependent RNA polymerase.     

TERRA介导的端粒维持

真核细胞线状染色体末端特殊结构被称为端粒,而端粒维持对于生命体来说具有十分重要的意义,其维持机制也十分复杂.端粒酶可以通过其具有的特殊逆转录酶特性,利用自身的RNA模板(TERC)以及具有催化功能的蛋白质亚基(TERT)延长端粒,维持其长度.本文着重综述端粒TERRA (telomeric repeat-contain...     

De novo telomere addition by Tetrahymena telomerase in vitro.

Previous molecular genetic studies have shown that during programmed chromosomal healing, telomerase adds telomeric repeats directly to non-telomeric sequences in Tetrahymena, forming de novo telomeres. However, the biochemical mechanism underlying this process is not well understood. Here, we show for the first time that telomerase activity is capable in vitro of efficiently elongating completely non-telomeric DNA oligonucleotide primers, consisting of natural telomere-adjacent or random sequences, at low primer concentrations. Telomerase activity isolated from mated or vegetative cells had indistinguishable specificities for nontelomeric and telomeric primers. Consistent with in vivo results, the sequence GGGGT... was the predominant initial DNA sequence added by telomerase in vitro onto the 3' end of the non-telomeric primers. The 3' and 5' sequences of the primer both influenced the efficiency and pattern of de novo telomeric DNA addition. Priming of telomerase by double-stranded primers with overhangs of various lengths showed a requirement for a minimal 3' overhang of 20 nucleotides. With fully single-stranded non-telomeric primers, primer length up to approximately 30 nucleotides strongly affected the efficiency of telomeric DNA addition. We propose a model for the primer binding site of telomerase for non-telomeric primers to account for these length and structural requirements. We also propose that programmed de novo telomere addition in vivo is achieved through a hitherto undetected intrinsic ability of telomerase to elongate completely non-telomeric sequences.     

Insights into the RNA quadruplex binding specificity of DDX21

Guanine quadruplexes can form in both DNA and RNA and influence many biological processes through various protein interactions. The DEAD-box RNA helicase protein DDX21 has been shown to bind and remodel RNA quadruplexes but little is known about its specificity for different quadruplex species. Previous reports have suggested DDX21 may interact with telomeric repeat containing RNA quadruplex (TERRA), an integral component of the telomere that contributes to telomeric heterochromatin formation and telomere length regulation. Here we report that the C-terminus of DDX21 directly interacts with TERRA. We use, for the first time, 2D saturation transfer difference NMR to map the protein binding site on a ribonucleic acid species and show that the quadruplex binding domain of DDX21 interacts primarily with the phosphoribose backbone of quadruplexes. Furthermore, by mutating the 2′OH of loop nucleotides we can drastically reduce DDX21's affinity for quadruplex, indicating that the recognition of quadruplex and specificity for TERRA is mediated by interactions with the 2′OH of loop nucleotides.