Identification of two conserved aspartic acid residues required for DNA digestion by a novel thermophilic Exonuclease VII in Thermotoga maritima

Exonuclease VII was first identified in 1974 as a DNA exonuclease that did not require any divalent cations for activity. Indeed, Escherichia coli ExoVII was identified in partially purified extracts in the presence of EDTA. ExoVII is comprised of two subunits (XseA and XseB) that are highly conserved and present in most sequenced prokaryotic genomes, but are not seen in eukaryotes. To better understand this exonuclease family, we have characterized an ExoVII homolog from Thermotoga maritima. Thermotoga maritima XseA/B homologs TM1768 and TM1769 were co-expressed and purified, and show robust nuclease activity at 80°C. This activity is magnesium dependent and is inhibited by phosphate ions, which distinguish it from E. coli ExoVII. Nevertheless, both E. coli and T. maritima ExoVII share a similar putative active site motif with two conserved aspartate residues in the large (XseA/TM1768) subunit. We show that these residues, Asp235 and Asp240, are essential for the nuclease activity of T. maritima ExoVII. We hypothesize that the ExoVII family of nucleases can be sub-divided into two sub-families based on EDTA resistance and that T. maritima ExoVII is the first member of the branch that is characterized by EDTA sensitivity and inhibition by phosphate

The effect of the TRF2 N-terminal and TRFH regions on telomeric G-quadruplex structures

The sequence of human telomeric DNA consists of tandem repeats of 5′-d(TTAGGG)-3′. This guanine-rich 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 (TRF2B), as well as this region plus the TRFH domain of TRF2 (TRF2BH), 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+. TRF2B stimulated the formation of parallel-stranded structures and, in some cases, intermolecular structures. TRF2BH also stimulated intermolecular but not parallel-stranded structures. Only full-length TRF2 and TRF2BH 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

The Telomere Binding Protein TRF2 Induces Chromatin Compaction

Mammalian telomeres are specialized chromatin structures that require the telomere binding protein, TRF2, for maintaining chromosome stability. In addition to its ability to modulate DNA repair activities, TRF2 also has direct effects on DNA structure and topology. Given that mammalian telomeric chromatin includes nucleosomes, we investigated the effect of this protein on chromatin structure. TRF2 bound to reconstituted telomeric nucleosomal fibers through both its basic N-terminus and its C-terminal DNA binding domain. Analytical agarose gel electrophoresis (AAGE) studies showed that TRF2 promoted the folding of nucleosomal arrays into more compact structures by neutralizing negative surface charge. A construct containing the N-terminal and TRFH domains together altered the charge and radius of nucleosomal arrays similarly to full-length TRF2 suggesting that TRF2-driven changes in global chromatin structure were largely due to these regions. However, the most compact chromatin structures were induced by the isolated basic N-terminal region, as judged by both AAGE and atomic force microscopy. Although the N-terminal region condensed nucleosomal array fibers, the TRFH domain, known to alter DNA topology, was required for stimulation of a strand invasion-like reaction with nucleosomal arrays. Optimal strand invasion also required the C-terminal DNA binding domain. Furthermore, the reaction was not stimulated on linear histone-free DNA. Our data suggest that nucleosomal chromatin has the ability to facilitate this activity of TRF2 which is thought to be involved in stabilizing looped telomere structures

The Outer Limits: Telomere maintenance by TRF2 and G-quadruplex DNA structures

Human telomeric DNA consists of tandem repeats of the sequence 5\u27-d(TTAGGG)-3\u27 assembled into a nucleoprotein complex that functions to protect the ends of chromosomes. Such guanine-rich DNA is capable of forming a variety of G-quadruplexes, which in turn, can have varying functional consequences on telomere maintenance. G-quadruplex stabilizing ligands have been shown to induce chromosome end-to-end fusions, senescence and apoptosis, effects similar to the expression of a dominant-negative TTAGGG Repeat Factor 2 (TRF2). With this in mind, we analyzed the effect of sequence and length of human telomeric DNA, as well as cation conditions on G-quadruplex formation by native polyacrylamide gel electrophoresis and circular dichroism. Although the structures of short telomeric oligonucleotides have been carefully examined, few studies have focused on longer telomeric DNA, which mimics the very ends of telomeres. We show that K + and Sr2+ can induce human telomeric DNA to form both inter- and intramolecular structures. Circular dichroism results suggest that the structures in K+ were a mix of parallel and antiparallel G-quadruplexes, while Sr2+ induced only parallel-stranded structures. We also found that TRF2, a protein essential for telomere maintenance, affects G-quadruplex structure. These structures serve as useful models to study the effects of G-quadruplexes on the activities of telomeric proteins, like TRF2, from human cells.The G-strand overhang at the ends of telomeres may periodically adopt at least some of these quadruplex conformations, which could subsequently affect protein binding and telomere function. TRF2, which binds towards the end of the double-strand (ds) telomere region just upstream of the G-strand overhang, is considered one of the key proteins in telomere protection and regulation. While TRF2 is not known to bind single-strand (ss) DNA, work performed in the lab suggested that the type of 3\u27-overhang in telomeric DNA ss/ds-junctions affects TRF2-binding. Specifically, preventing G-quadruplex formation by changing the overhang sequence from 5\u27-d(TTAGGG)4-3\u27, to 5\u27-dTTAGGG(TTAG AG)2TTAGGG-3\u27, reduced TRF2 recruitment to the ss/ds-junction from HeLa cell extracts. Using the same techniques as above, we show that the N-terminal basic domain of TRF2 in K+ induces a switch from the mixed parallel/antiparallel-stranded G-quadruplexes usually stabilized by K+-alone, to parallel-stranded G-quadruplexcs. Interestingly, it also promotes intermolecular parallel G-quadruplex formation on non-quadruplex, single-stranded intermediates, but will not induce a switch from an antiparallel to a parallel G-quadruplex in Na+. These results are the first to demonstrate specific TRF2-G-quadruplex interactions. They suggest a novel mechanism for TRF2 recognition of the double-strand/single-strand junction of telomeres, where the myb-like domain binds to the double-strand DNA and the N-terminal basic domain interacts with the overhang, stabilizing the interaction. This model has implications for TRF2 communication with the very terminus of the telomere and for stimulation of strand invasion proposed to stabilize t-loop structures

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