Flexibility of telomerase in binding the RNA template and DNA telomeric repeat

Telomerase synthesizes telomeres at the ends of linear chromosomes by repeated reverse transcription from a short RNA template. Crystal structures of Tribolium castaneum telomerase reverse transcriptase (tcTERT) and cryoelectron microscopy (cryo-EM) structures of human and Tetrahymena telomerase have revealed conserved features in the reverse-transcriptase domain, including a cavity near the DNA 3′ end and snug interactions with the RNA template. For the RNA template to translocate, it needs to be unpaired and separated from the DNA product. Here we investigate the potential of the structural cavity to accommodate a looped-out DNA bulge and enable the separation of the RNA/DNA hybrid. Using tcTERT as a model system, we show that a looped-out telomeric repeat in the DNA primer can be accommodated and extended by tcTERT but not by retroviral reverse transcriptase. Mutations that reduce the cavity size reduce the ability of tcTERT to extend the looped-out DNA substrate. In agreement with cryo-EM structures of telomerases, we find that tcTERT requires a minimum of 4 bp between the RNA template and DNA primer for efficient DNA synthesis. We also have determined the ternary-complex structure of tcTERT including a downstream RNA/DNA hybrid at 2.0-Å resolution and shown that a downstream RNA duplex, equivalent to the 5′ template-boundary element in telomerase RNA, enhances the efficiency of telomere synthesis by tcTERT. Although TERT has a preformed active site without the open-and-closed conformational changes, it contains cavities to accommodate looped-out RNA and DNA. The flexible RNA–DNA binding likely underlies the processivity of telomeric repeat addition.

Expression of the Reverse Transcriptase Domain of Telomerase Reverse Transcriptase Induces Lytic Cellular Response in DNA-Immunized Mice and Limits Tumorigenic and Metastatic Potential of Murine Adenocarcinoma 4T1 Cells

Telomerase reverse transcriptase (TERT) is a classic tumor-associated antigen overexpressed in majority of tumors. Several TERT-based cancer vaccines are currently in clinical trials, but immune correlates of their antitumor activity remain largely unknown. Here, we characterized fine specificity and lytic potential of immune response against rat TERT in mice. BALB/c mice were primed with plasmids encoding expression-optimized hemagglutinin-tagged or nontagged TERT or empty vector and boosted with same DNA mixed with plasmid encoding firefly luciferase (Luc DNA). Injections were followed by electroporation. Photon emission from booster sites was assessed by in vivo bioluminescent imaging. Two weeks post boost, mice were sacrificed and assessed for IFN-γ, interleukin-2 (IL-2), and tumor necrosis factor alpha (TNF-α) production by T-cells upon their stimulation with TERT peptides and for anti-TERT antibodies. All TERT DNA-immunized mice developed cellular and antibody response against epitopes at the N-terminus and reverse transcriptase domain (rtTERT) of TERT. Photon emission from mice boosted with TERT/TERT-HA+Luc DNA was 100 times lower than from vector+Luc DNA-boosted controls. Bioluminescence loss correlated with percent of IFN-γ/IL-2/TNF-α producing CD8+ and CD4+ T-cells specific to rtTERT, indicating immune clearance of TERT/Luc-coexpressing cells. We made murine adenocarcinoma 4T1luc2 cells to express rtTERT by lentiviral transduction. Expression of rtTERT significantly reduced the capacity of 4T1luc2 to form tumors and metastasize in mice, while not affecting in vitro growth. Mice which rejected the tumors developed T-cell response against rtTERT and low/no response to the autoepitope of TERT. This advances rtTERT as key component of TERT-based therapeutic vaccines against cancer.

THE EXISTENCE OF GENE DNA POLYMERASE MUTATION FROM POSITIVE HEPATITIS B SAMPLES IN BANDUNG, INDONESIA

Objective: To complete mutation existence in reverse transcriptase domain of the viral Polymerase.Methods: The study was done by amplification step of viral polymerase gene fragment, agarose gel electrophoresis, PCR product purification with GFX column kit, sequencing and sequencing result analysis. The samples were derived from Clinical Laboratory in Bandung Indonesia.Results: The result showed mutations in DNA fragment encoding for RT domain viral polymerase in sample 6, 7 and 8. There were mutations leading to amino acid substitution L526S in sample 6, D551E in sample 7 and D552E in sample 8.Conclusion: D551E and D552E substitution occurred in YMDD motif RT DNA polymerase that produced YMDE mutant. L526S, D551E and D552E were estimated as antivirus-resistance mutants that have never been reported before.

The yeast telomerase RNA, TLC1, participates in two distinct modes of TLC1-TLC1 association processesin vivo

Telomerase core enzyme minimally consists of the telomerase reverse transcriptase domain-containing protein (Est2 in budding yeastS. cerevisiae) and telomerase RNA, which contains the template specifying the telomeric repeat sequence synthesized. Here we report thatin vivo, a fraction ofS. cerevisiaetelomerase RNA (TLC1) molecules form complexes containing at least two molecules of TLC1, via two separable modes: one requiring a sequence in the 3′ region of the immature TLC1 precursor and the other requiring Ku and Sir4. Such physical TLC1-TLC1 association peaked in G1 phase and did not require telomere silencing, telomere tethering to the nuclear periphery, telomerase holoenzyme assembly, or detectable Est2-Est2 protein association. These data indicate that TLC1-TLC1 associations reflect processes occurring during telomerase biogenesis; we propose that TLC1-TLC1 associations and subsequent reorganization may be regulatory steps in telomerase enzymatic activation.

Bioinformatic analysis of Arabidopsis reverse transcriptases with a zinc-finger domain

Plants, as most eukaryotic organisms, harbor several genes encoding a reverse transcriptase domain. The majority of them are part of transposable elements (TEs) and/or retroviral genomes that have been inserted into their genomes. However, there are some examples of RT domain-containing genes that have been endogenized during plant evolution; these genes appear to display functions other than “selfish” maintenance and replication of TEs, and subjected to host gene regulation. In the present work we have analyzed a subset of genes in Arabidopsis with an RT domain (RVT) containing a zinc finger motif (Znf), termed RVT-Znf domain, with structural characteristics of endogenous genes i.e., contain potential upstream regions as well as 5’UTR, and 3’UTR, and are not flanked by retroelement features. Phylogenetic analysis of these genes, based on the RVT-Znf domain, indicates that there are three clades, the members of which having additional domains. When compared to additional sequences, RVT-Znf formed a cluster that is more closely related to non-LTR retrotransposons and group II introns. Extant data from microarray databases indicate that several Arabidopsis genes are expressed. These data indicate that these RTs may have been endogenized. Possible roles for these genes are discussed.

The yeast telomerase RNA, TLC1, participates in two distinct modes of TLC1-TLC1 association processes in vivo

Telomerase core enzyme minimally consists of the telomerase reverse transcriptase domain-containing protein (Est2 in budding yeast S. cerevisiae) and telomerase RNA, which contains the template specifying the telomeric repeat sequence synthesized. Here we report that in vivo, a fraction of S. cerevisiae telomerase RNA (TLC1) molecules form complexes containing at least two molecules of TLC1, via two separable modes: one requiring a sequence in the 3’ region of the immature TLC1 precursor and the other requiring Ku and Sir4. Such physical TLC1-TLC1 association peaked in G1 phase and did not require telomere silencing, telomere tethering to the nuclear periphery, telomerase holoenzyme assembly, or detectable Est2-Est2 protein association. These data indicate that TLC1-TLC1 associations reflect processes occurring during telomerase biogenesis; we propose that TLC1-TLC1 associations and subsequent reorganization may be regulatory steps in telomerase enzymatic activation.