G4 motifs are greatly enriched near promoters, suggesting that quadruplex structures may be targets of transcriptional regulation. Here we show, by ChIP-Seq analysis of human cells, that 40% of the binding sites of the transcription-associated helicases, XPB and XPD, overlap with G4 motifs. The highly significant overlap of XPB and XPD binding sites with G4 motifs cannot be explained by GC-richness or parameters of the genomewide analysis, but instead suggests that these proteins are recruited to quadruplex structures that form in genomic DNA (G4 DNA). Biochemical analysis demonstrates that XPD is a robust G4 DNA helicase, and XPB binds to G4 DNA. XPB and XPD are enriched near the transcription start site (TSS) at 20% of genes, especially highly transcribed genes. XPB and XPD enrichment at G4 motifs characterizes specific signaling pathways and regulatory pathways associated with specific cancers. These results identify new candidate pathways for therapies targeted to quadruplexes.

Key antigens of Leishmania species identified in the context of host responses in Leishmania-exposed individuals from disease-endemic areas were prioritized for the development of a subunit vaccine against visceral leishmaniasis (VL), the most deadly form of leishmaniasis. Two Leishmania proteins—nucleoside hydrolase and a sterol 24-c-methyltransferase, each of which are protective in animal models of VL when properly adjuvanted— were produced as a single recombinant fusion protein NS (LEISH-F3) for ease of antigen production and broad coverage of a heterogeneous major histocompatibility complex population. When formulated with glucopyranosyl lipid A-stable oil-in-water nanoemulsion (GLA-SE), a Toll-like receptor 4 TH1 (T helper 1) promoting nanoemulsion adjuvant, the LEISH-F3 polyprotein induced potent protection against both L. donovani and L. infantum in mice, measured as significant reductions in liver parasite burdens. A robust immune response to each component of the vaccine with polyfunctional CD4 TH1 cell responses characterized by production of antigen-specific interferon-γ, tumor necrosis factor and interleukin-2 (IL-2), and low levels of IL-5 and IL-10 was induced in immunized mice. We also demonstrate that CD4 T cells, but not CD8 T cells, are sufficient for protection against L. donovani infection in immunized mice. Based on the sum of preclinical data, we prepared GMP materials and performed a phase 1 clinical study with LEISH-F3+GLA-SE in healthy, uninfected adults in the United States. The vaccine candidate was shown to be safe and induced a strong antigen-specific immune response, as evidenced by cytokine and immunoglobulin subclass data. These data provide a strong rationale for additional trials in Leishmania-endemic countries in populations vulnerable to VL.

The RNA Pol II transcription complex pauses just downstream of the promoter in a significant fraction of human genes. The local features of genomic structure that contribute to pausing have not been defined. Here, we show that genes that pause are more G-rich within the region flanking the transcription start site (TSS) than RefSeq genes or non-paused genes. We show that enrichment of binding motifs for common transcription factors, such as SP1, may account for G-richness upstream but not downstream of the TSS. We further show that pausing correlates with the presence of a GrIn1 element, an element bearing one or more G4 motifs at the 5′-end of the first intron, on the non-template DNA strand. These results suggest potential roles for dynamic G4 DNA and G4 RNA structures in cis-regulation of pausing, and thus genome-wide regulation of gene expression, in human cells.

Sustained elimination of Visceral Leishmaniasis (VL) requires the reduction and control of parasite reservoirs to minimize the transmission of Leishmania donovani infection. A simple, reproducible and definitive diagnostic procedure is therefore indispensable for the early and accurate detection of parasites in VL, Relapsed VL (RVL) and Post Kala-azar Dermal Leishmaniasis (PKDL) patients, all of whom are potential reservoirs of Leishmania parasites. To overcome the limitations of current diagnostic approaches, a novel quantitative real-time polymerase chain reaction (qPCR) method based on Taqman chemistry was devised for the detection and quantification of L. donovani in blood and skin. The diagnostic efficacy was evaluated using archived peripheral blood buffy coat DNA from 40 VL, 40 PKDL, 10 RVL, 20 cured VL, and 40 cured PKDL along with 10 tuberculosis (TB) cases and 80 healthy endemic controls. Results were compared to those obtained using a Leishmania-specific nested PCR (Ln-PCR). The real time PCR assay was 100% (95% CI, 91.19–100%) sensitive in detecting parasite genomes in VL and RVL samples and 85.0% (95% CI, 70.16–94.29%) sensitive for PKDL samples. In contrast, the sensitivity of Ln-PCR was 77.5% (95% CI, 61.55–89.16%) for VL samples, 100% (95%CI, 69.15–100%) for RVL samples, and 52.5% (95% CI, 36.13–68.49%) for PKDL samples. There was significant discordance between the two methods with the overall sensitivity of the qPCR assay being considerably higher than Ln-PCR. None of the assay detected L. donovani DNA in buffy coats from cured VL cases, and reduced infectious burdens were demonstrated in cured PKDL cases who remained positive in 7.5% (3/40) and 2.5% (1/40) cases by real-time PCR and Ln-PCR, respectively. Both assays were 100% (95% CI, 95.98–100) specific with no positive signals in either endemic healthy control or TB samples. The real time PCR assay we developed offers a molecular tool for accurate detection of circulating L. donovani parasites in VL, PKDL and RVL patients, as well as being capable of assessing response to treatment. As such, this real time PCR assay represents an important contribution in efforts to eliminate VL.

Visceral leishmaniasis (VL) in South Asia is a serious disease affecting children and adults. Acute VL develops in only a fraction of those infected individuals, the majority being asymptomatic with the potential to transmit infection and develop disease. We followed 56 individuals characterized as being asymptomatic by sero-positivity with rk39 RDT in a hyper endemic district of Bangladesh to define the utility of Leishmania-specific antibodies and DNA in identifying infection. At baseline, 54 of the individuals were sero-positive with one or more quantitative antibody assays and antibody levels persisted at follow-up. Most sero-positive individuals (47/54) tested positive by qPCR at baseline, but only 16 tested positive at follow-up. The discrepancies among the different tests may shed light on the dynamics of asymptomatic infections of L. donovani, as well as underscore the need for standard diagnostic tools for active surveillance as well as assessing the effectiveness of prophylactic and therapeutic interventions.

BackgroundVisceral leishmaniasis (VL) can be fatal without timely diagnosis and treatment. Treatment efficacies vary due to drug resistance, drug toxicity and co-morbidities. It is important to monitor treatment responsiveness to confirm cure and curtail relapse. Currently, microscopy of spleen, bone marrow or lymph node biopsies is the only definitive method to evaluate cure. A less invasive test for treatment success is a high priority for VL management.MethodsIn this study, we describe the development of a capture ELISA based on detecting Leishmania donovani antigens in urine samples and comparison with the Leishmania Antigen ELISA, also developed for the same purpose. Both were developed as prototype kits and tested on patient urine samples from Sudan, Ethiopia, Bangladesh and Brazil, along with appropriate control samples from endemic and non-endemic regions. Sensitivity and specificity were assessed based on accurate detection of patients compared to control samples. One- Way ANOVA was used to assess the discrimination capacity of the tests and Cohen’s kappa was used to assess their correlation.ResultsThe Leishmania Antigen Detect™ ELISA demonstrated >90 % sensitivity on VL patient samples from Sudan, Bangladesh and Ethiopia and 88 % on samples from Brazil. The Leishmania Antigen ELISA was comparable in performance except for lower sensitivity on Sudanese samples. Both were highly specific. To confirm utility in monitoring treatment, urine samples were collected from VL patients at days 0, 30 and 180 post- treatment. For the Leishmania Antigen Detect™ ELISA, positivity was high at day 0 at 95 %, falling to 21 % at day 30. At day 180, all samples were negative, corresponding well with clinical cure. A similar trend was also seen for the Leishmania Antigen ELISA albeit; with lower positivity of 91 % at Day 0 and more patients, remaining positive at Days 30 and 180.DiscussionThe Leishmania Antigen Detect™ and the Leishmania Antigen ELISAs are standardized, user- friendly, quantitative and direct tests to detect Leishmania during acute VL as well as to monitor parasite clearance during treatment. They are a clear improvement over existing options.ConclusionThe ELISAs provide a non-invasive method to detect parasite antigens during acute infection and monitor its clearance upon cure, filling an unmet need in VL management. Further refinement of the tests with more samples from endemic regions will define their utility in monitoring treatment.

Human alkyladenine DNA glycosylase "flips" damaged DNA bases into its active site where excision occurs. Tyrosine 162 is inserted into the DNA helix in place of the damaged base and may assist in nucleotide flipping by "pushing" it. Mutating this DNA-intercalating Tyr to Ser reduces the DNA binding and base excision activities of alkyladenine DNA glycosylase to undetectable levels demonstrating that Tyr-162 is critical for both activities. Mutation of Tyr-162 to Phe reduces the single turnover excision rate of hypoxanthine by a factor of 4 when paired with thymine. Interestingly, when the base pairing partner for hypoxanthine is changed to difluorotoluene, which cannot hydrogen bond to hypoxanthine, single turnover excision rates increase by a factor of 2 for the wild type enzyme and about 3 to 4 for the Phe mutant. In assays with DNA substrates containing 1,N 6 -ethenoadenine, which does not form hydrogen bonds with either thymine or difluorotoluene, base excision rates for both the wild type and Phe mutant were unaffected. These results are consistent with a role for Tyr-162 in pushing the damaged base to assist in nucleotide flipping and indicate that a nucleotide flipping step may be rate-limiting for excision of hypoxanthine.Human alkyladenine DNA glycosylase (AAG) 1 is one of several damage-specific DNA glycosylases that function in the base excision repair pathway (reviewed in Refs. 1-4). These DNA glycosylases initiate repair by identifying and removing damaged bases from DNA. Monofunctional DNA glycosylases, including AAG, hydrolyze the glycosylic bond between the base and sugar to leave an abasic sugar residue in DNA. Other enzymes in the pathway remove this apurinic/apyrimidinic lesion and resynthesize DNA to complete repair. The ability of DNA glycosylases to identify and excise damaged DNA bases is key to the overall success of base excision repair.Structural studies of AAG (5, 6) and other DNA glycosylases have revealed that they use a nucleotide "flipping" mechanism for damaged base recognition and excision where the damaged base is flipped out of the DNA helix and bound in an enzyme active site. In these nucleotide-flipped DNA glycosylase⅐DNA complexes, an enzyme amino acid side chain is inserted into the base stack at the site vacated by the flipped base and may assist in nucleotide flipping by pushing the damaged base from the helix. It is believed that DNA glycosylases actively flip damaged bases out of the helix rather than passively capturing bases that have transiently adopted extrahelical conformations. This active nucleotide flipping mechanism is supported by detailed kinetic studies of Escherichia coli uracil DNA glycosylase which show a two-step binding mechanism where UDG initially binds DNA to form an unflipped protein⅐DNA complex prior to flipping uracil from the helix (7).Many questions remain about how nucleotide flipping enables DNA glycosylases to discriminate between damaged and undamaged bases. For DNA glycosylases that have a narrow substrate specificity, a mechanism where a...

Eukaryotic exonuclease 1 functions in replication, recombination, mismatch repair, telomere maintenance, immunoglobulin (Ig) gene class switch recombination, and somatic hypermutation. The enzyme has 5-3 exonuclease, flap endonuclease, and weak RNaseH activity in vitro, but it has been difficult to reconcile these activities with its diverse biological functions. We report robust cleavage by human exonuclease 1 of transcribed G-rich DNA sequences with potential to form G loops and G4 DNA. Predicted Ig switch recombination intermediates are substrates for both exonucleolytic and 5 flap endonucleolytic cleavage. Excision is nick-dependent and structure-dependent. These results lead to a model for exonuclease 1 function in class switch recombination in which cleavage at activation-induced deaminase (AID)-initiated nicks produces gaps that become substrates for further attack by AID and subsequent repair.antibody ͉ B cell ͉ cotranscriptional hybrid ͉ G4 DNA ͉ switch recombination E ukaryotic exonuclease 1 (EXO1) belongs to class III of the RAD2 family of nucleases, which includes structure-specific nucleases like flap endonuclease 1 (FEN1). Purified recombinant human EXO1 (hEXO1) possesses 5Ј-3Ј exonuclease activity on dsDNA, 5Ј flap endonuclease activity, and weak RNaseH activity, and it is essential for both 5Ј and 3Ј nick-directed mismatch repair (1-4). In Saccharomyces cerevisiae, EXO1 functions in mismatch repair, replication, recombination, and at the telomeres (5-8). Mammalian EXO1 was identified as a homolog of S. cerevisiae Exo1p (1, 9) and by virtue of its interaction with hMSH2 (10). hEXO1 functionally complements its yeast homolog, and hEXO1 overexpression rescues conditional lethality of S. cerevisiae rad27 (FEN1) mutants (2), suggesting some overlap of functions of EXO1 and FEN1. Exo1 Ϫ/Ϫ mice display impaired meiosis, microsatellite instability, increased level of mutations and frequency of lymphomas (11), diminished efficiency of immunoglobulin (Ig) class switch recombination, and an altered spectrum of somatic hypermutation (12). In mice with dysfunctional telomeres, EXO1-deficiency extends life span and reduces genomic instability (13).Class switch recombination is a regulated process of DNA deletion that joins a new constant region to the rearranged and expressed Ig heavy chain variable region in activated B cells (14-16). Recombination junctions are within switch (S) regions, 2-to 10-kb noncoding regions that are transcribed from a dedicated upstream promoter to activate recombination (Fig. 1A). Recombination is initiated by C to U deamination by the B cell-specific enzyme, activation-induced deaminase (AID), which preferentially deaminates within the consensus motif, WRCY (W ϭ A/T, R ϭ purine, Y ϭ pyrimidine). AID-initiated damage is processed by redundant pathways, dependent on either uracil excision by UNG or mismatch recognition by MutS␣ (17). DNA nicking may then be promoted by the MRE11 AP lyase (18) or other AP endonucleases and lyases (19) or by factors that nick DNA for mismatch repair ...