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Get crazy with the wild erratic synth riffs and melody loops, 808s and distorted leads. Create hype with melodic fillers and risers. Infuse your track with some real tribal feels by playing around with the ethnic melody and tribal percussion loops. Smash the dancefloor to shreds with our heavy drag and drop drums and bring some creativity and life into your tracks with the dope vocal chants and shouts included in this pack. This complete collection will dazzle your crowd and make it know you are a force to be reckoned with!

While RAD51 is likely to catalyze strand invasion of TERRA into telomeric DNA, several telomere-associated proteins have also been identified to regulate strand invasion and telomere retention (Figure 1). Among the shelterin components, TRF1, TRF2 and POT1 play critical roles. The N-terminal basic domain of TRF2 can bind TERRA and stimulate R-loop formation in vitro [37]. This activity is prevented in vitro and in vivo by TRF1 through its N-terminal acidic domain. Whether TRF2 promotes R-loops under physiological conditions is not known, though TRF2 depletion per se does not decrease R-loop formation at telomeres [34,37]. POT1 binds specifically to the single-stranded G-rich telomeric DNA but not to the corresponding 5ʹ-UUAGGG-3ʹ repeats in TERRA [38]. POT1 deletion in human cells causes rapidly dramatic telomere elongation by RAD51-mediated homologous recombination [39]. At the same time the telomeric R-loops are increased. It is unclear if TERRA R-loops are formed as an immediate consequence of POT1-loss and if they are a prerequisite for telomere recombination. Perhaps more likely, POT1-loss liberates the single-stranded telomeric DNA for RPA binding which subsequently, with assistance of BRCA2, becomes replaced by RAD51. The increased local concentration of RAD51 may facilitate the binding of TERRA by RAD51 in a succeeding step, which will trigger strand invasion and R-loop formation. The DNA strand that is displaced by the TERRA R-loop may recruit additional RPA and RAD51 in a feedforward loop to first promote RPA-dependent DNA checkpoint signaling and subsequently replacement by RAD51 to mediate homologous recombination (Figure 4).

The FANCM protein, whose mutation has been associated with Fanconi Anemia, is an ATPase associated with DNA branch migration. Its roles at telomeres have been characterized in U2OS cells [40,41], which are ALT cells (for alternative lengthening of telomeres) that use DNA recombination to maintain telomeric DNA repeats. Strikingly, FANCM depletion caused an increase in R-loops both at telomeres and elsewhere in the genome. In addition, FANCM can resolve telomeric R-loops in vitro using its RNA:DNA helicase activity suggesting that it directly participates in R-loop resolution [40]. NONO/SFPQ heterodimers, which are involved in various aspects of RNA metabolism in the nucleus, have also been implicated in suppressing telomeric R-loops [42]. Their depletion increased R-loop signals partially colocalizing with telomeres in nuclei of U2OS cells. Most recently, the RTEL1 helicase has been implicated in TERRA regulation [43]. RTEL1 has well-established crucial roles during telomere replication resolving T-loops in S phase as well as telomeric G-quadruplex structures [44]. The new work shows that RTEL1 also binds in vitro TERRA 5ʹ-UUAGGG-3ʹ repeats when adopting a G-quadruplex structure [43]. Strikingly, RTEL1 deletion caused strong increase in TERRA levels, while TERRA association with telomeres was significantly diminished. It was proposed that RTEL1 facilitates TERRA association with chromosome ends through a stimulatory effect on telomeric R-loops, which in turn could prevent additional transcription from the R-loop containing telomeres [43]. This proposed mode of action deviates from the documented roles of RTEL1 at G-quadruplex forming DNA sequences elsewhere in the genome where RTEL1 dismantles R-loops [45]. Therefore, it will be interesting to further dissect the mechanism and also test if and to what extent the S phase-specific telomere recruitment of RTEL1 by dephosphorylated TRF2 may contribute [46]. Certainly, also other models could be at play. For example, if RTEL1 affected TERRA 3ʹ end formation preventing its polyadenylation, absence of RTEL1 and TERRA polyadenylation might also stabilize this RNA and trigger its dissociation from chromosome ends [24].

While break-induced replication of telomeres can proceed through Rad51-dependent and independent mechanisms in yeast [61], the different synthesis pathways which operate in human ALT cells have not been fully dissected. Several studies documented roles of RAD51 in ALT cells. Depletion of RAD51 decreased the formation of ALT-associated promyelocytic leukemia bodies (APBs) in ALT cells in which telomeres gather for ALT DNA synthesis [62]. Also, chemical inhibition of RAD51 in ALT cells interfered with telomere maintenance [63] and RAD51 depletion with the frequency of telomere extension events [64]. In addition, human RAD51 facilitates long-range telomere movement and telomere clustering in ALT cells [65]. On the other hand, based on RAD51-depletion experiments, other studies described RAD51-independent mechanisms for break-induced telomere synthesis in ALT cells. A major mechanism involves RAD52, while a second ill-defined mechanism can be observed in the absence of RAD52 [54,66]. Remarkably, RAD52 can bind RNA:DNA duplexes [67,68] as well as single and double stranded DNA and thus may associate with TERRA R-loops to mediate a PCNA-RFC-Polδ-dependent pathway for conservative DNA synthesis that extends telomere length. Overall, the published work demonstrates that distinct mechanisms contribute to telomere elongation in human ALT cells. In our view, however, it may be premature to exclude crucial roles of RAD51 in a subset of the mechanisms, as this protein is required for cell viability and essential functions may have been retained in the knockdown studies.

R-loops had been originally considered as toxic by-products of transcription which generate obstacles for DNA and RNA polymerases, inducing DNA damage upon replication [69,70]. Also at telomeres, R-loops do interfere with the semiconservative DNA replication machinery. The THO complex, which has been implicated in removing nascent RNA from chromatin, is present at telomeres in human cells and in budding yeast [4,71]. Deletion of THO components in S. cerevisiae increases telomeric R-loops, leading to replication stress at telomeres and telomere shortening [71]. As discussed above, RNase H enzymes also counteract R-loops. In their absence the accumulation of telomeric R-loops leads to telomere loss and accelerated senescence in recombination-deficient budding yeast, supporting the notion that R-loops interfere with telomere replication [72]. Finally, depletion of NMD factors in human cells led to increased TERRA at telomeres and frequent loss of telomeric DNA [10]. Specifically, UPF1 depletion leads to inefficient replication of telomeres synthesized by leading strand synthesis, suggesting a role of this helicase in removing TERRA from leading strand telomeres for their replication [73].

Hypomorphic mutations in the de novo DNA methyltransferase DNMT3b cause ICF (Immunodeficiency, Centromeric instability and Facial anomalies) syndrome type I [27]. Subtelomeric DNA sequences are hypomethylated in ICF type I syndrome cells, correlating with strongly increased TERRA levels. These cells also display increased telomeric R-loops throughout the cell cycle, telomere damage, accelerated telomere shortening and premature replicative senescence, all being consistent with the interference of TERRA R-loops with telomere replication [74]. However, a direct demonstration of causative roles of DNMT3b deficiency and subtelomeric DNA demethylation in the short telomere phenotype has been cumbersome. The DNA methylation status at subtelomeres cannot be rescued in the cellular ICF models with ectopic DNMT3b, indicative of a persistent epigenetic memory [75].

Here, we sought to identify a panel of trimers to immunofocus V2 and FP epitopes. Specifically, we sought well-expressed trimers with a range of CH01 sensitivities from at least 5 diverse strains to enable us to test various prime-boost vaccine concepts. Strains with broad sensitivity to multiple V2 NAbs are preferred. Ideally, the panel should encompass a range of NAb sensitivities, ranging from acute, UCA-triggering to that of typical transmitted isolates. All selected trimers should be functional in pseudovirus (PV) assays, so their NAb sensitivity can be calibrated and should not be overtly sensitive to V3 MAbs.

We plan to use CH01 UCA-sensitive and FP-sensitive mutants to prime, followed by boosts with a variety of strains and/or mutants to increase stringency and promote binding to anchor residues and to tolerate sequence variation and clashing glycans. Thus, strand C could become gradually more neutral, mimicking waves of diversity in V2 NAb ontogeny in natural infection [25,37,70,110]. In a vaccine setting, strain variation between shots could be helpful, provided that bNAbs stays on track. Overall, the diverse panel of membrane trimers described here will allow us to test a variety of vaccine concepts for immunofocusing V2 and FP NAbs.

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