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Ribonucleotides are the natural analogs of deoxyribonucleotides, which can be misinserted by DNA polymerases, leading to the most abundant DNA lesions in genomes. During replication, DNA polymerases tolerate patches of ribonucleotides on the parental strands to different extents. The majority of human DNA polymerases have been reported to misinsert ribonucleotides into genomes. However, only PrimPol, DNA polymerase α, telomerase, and the mitochondrial human DNA polymerase (hpol) γ have been shown to tolerate an entire RNA strand. Y-family hpol η is known for translesion synthesis opposite the UV-induced DNA lesion cyclobutane pyrimidine dimer and was recently found to incorporate ribonucleotides into DNA. Here, we report that hpol η is able to bind DNA/DNA, RNA/DNA, and DNA/RNA duplexes with similar affinities. In addition, hpol η, as well as another Y-family DNA polymerase, hpol κ, accommodates RNA as one of the two strands during primer extension, mainly by inserting dNMPs opposite unmodified templates or DNA lesions, such as 8-oxo-2'-deoxyguanosine or cyclobutane pyrimidine dimer, even in the presence of an equal amount of the DNA/DNA substrate. The discovery of this RNA-accommodating ability of hpol η redefines the traditional concept of human DNA polymerases and indicates potential new functions of hpol η .
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Recent progress in large-scale nucleic acid analysis technology has revealed the presence of vast numbers of RNA species in cells, and extensive processing. To investigate the functions of these transcripts highly efficient methods are needed to analyze their interactions with RNA-binding proteins (RNBPs), and to understand the binding mechanisms. Many methods have been described to identify RNBPs, but none are wholly satisfactory, in part because RNAs are flexible macromolecules that adopt multiple conformations only some of which might bind to specific proteins. Here we describe a novel in vitro RNA-pull-down assay using tRNA scaffolded Streptavidin Aptamer (tRSA), to identify transcript specific RNA binding protein from mammalian cell lysates. The tRNA scaffold functions to stabilize the structure of the aptamer and the attached RNA, increasing the efficiency of the affinity purification.
SMARCAL1 catalyzes replication fork remodeling to maintain genome stability. It is recruited to replication forks via an interaction with replication protein A (RPA), the major ssDNA-binding protein in eukaryotic cells. In addition to directing its localization, RPA also activates SMARCAL1 on some fork substrates but inhibits it on others, thereby conferring substrate specificity to SMARCAL1 fork-remodeling reactions. We investigated the mechanism by which RPA regulates SMARCAL1. Our results indicate that although an interaction between SMARCAL1 and RPA is essential for SMARCAL1 activation, the location of the interacting surface on RPA is not. Counterintuitively, high-affinity DNA binding of RPA DNA-binding domain (DBD) A and DBD-B near the fork junction makes it easier for SMARCAL1 to remodel the fork, which requires removing RPA. We also found that RPA DBD-C and DBD-D are not required for SMARCAL1 regulation. Thus, the orientation of the high-affinity RPA DBDs at forks dictates SMARCAL1 substrate specificity.
© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
Stapled helix peptides can serve as useful tools for inhibiting protein-protein interactions but can be difficult to optimize for affinity. Here we describe the discovery and optimization of a stapled helix peptide that binds to the N-terminal domain of the 70 kDa subunit of replication protein A (RPA70N). In addition to applying traditional optimization strategies, we employed a novel approach for efficiently designing peptides containing unnatural amino acids. We discovered hot spots in the target protein using a fragment-based screen, identified the amino acid that binds to the hot spot, and selected an unnatural amino acid to incorporate, based on the structure-activity relationships of small molecules that bind to this site. The resulting stapled helix peptide potently and selectively binds to RPA70N, does not disrupt ssDNA binding, and penetrates cells. This peptide may serve as a probe to explore the therapeutic potential of RPA70N inhibition in cancer.
Despite androgen deprivation therapy (ADT), persistent androgen receptor (AR) signaling enables outgrowth of castration resistant prostate cancer (CRPC). In prostate cancer (PCa) cells, ADT may enhance AR activity through induction of oxidative stress. Herein, we investigated the roles of Nrf1 and Nrf2, transcription factors that regulate antioxidant gene expression, on hormone-mediated AR transactivation using a syngeneic in vitro model of androgen dependent (LNCaP) and castration resistant (C4-2B) PCa cells. Dihydrotestosterone (DHT) stimulated transactivation of the androgen response element (ARE) was significantly greater in C4-2B cells than in LNCaP cells. DHT-induced AR transactivation was coupled with higher nuclear translocation of p65-Nrf1 in C4-2B cells, as compared to LNCaP cells. Conversely, DHT stimulation suppressed total Nrf2 levels in C4-2B cells but elevated total Nrf2 levels in LNCaP cells. Interestingly, siRNA mediated silencing of Nrf1 attenuated AR transactivation while p65-Nrf1 overexpression enhanced AR transactivation. Subsequent studies showed that Nrf1 physically interacts with AR and enhances AR's DNA-binding activity, suggesting that the p65-Nrf1 isoform is a potential AR coactivator. In contrast, Nrf2 suppressed AR-mediated transactivation by stimulating the nuclear accumulation of the p120-Nrf1 which suppressed AR transactivation. Quantitative RT-PCR studies further validated the inductive effects of p65-Nrf1 isoform on the androgen regulated genes, PSA and TMPRSS2. Therefore, our findings implicate differential roles of Nrf1 and Nrf2 in regulating AR transactivation in PCa cells. Our findings also indicate that the DHT-stimulated increase in p65-Nrf1 and the simultaneous suppression of both Nrf2 and p120-Nrf1 ultimately facilitates AR transactivation in CRPC cells.
Bacterial two-component systems (TCSs) mediate specific responses to distinct conditions and/or stresses. TCS interactions are highly specific between cognate partners to avoid unintended cross-talk. Although cross-talk between a sensor kinase and a noncognate response regulator has been previously demonstrated, the majority of reported interactions have not been robust. Here, we report that in the case of the quorum-sensing Escherichia coli (Qse)BC TCS, absence of the cognate sensor QseC leads to robust, constitutive activation of the QseB response regulator by the noncognate polymyxin resistance (Pmr) sensor kinase PmrB. Remarkably, the noncognate PmrB exhibits a kinetic preference for QseB that is similar to QseC. However, although PmrB readily phosphorylates QseB in vitro, it is significantly less efficient at dephosphorylating QseB, compared with QseC, thereby explaining the increased levels of active QseB in the qseC mutant. In addition to PmrB activating QseB on the protein level, we found that the PmrA response regulator contributes to qseB transcription in the absence of QseC and PmrA specifically binds the qseBC promoter, indicative of a direct regulation of qseBC gene transcription by PmrAB under physiological conditions. Addition of ferric iron in the growth medium of wild-type uropathogenic E. coli induced the expression of qseBC in a PmrB-dependent manner. Taken together, our findings suggest that (i) robust cross-talk between noncognate partners is possible and (ii) this interaction can be manipulated for the development of antivirulence strategies aimed at targeting uropathogenic Escherichia coli and potentially other QseBC-PmrAB-bearing pathogens.
The mammalian target of rapamycin (mTOR), an essential serine/threonine kinase, functions in biochemically distinct multiprotein complexes, but little is known about roles of the complexes in B cells. The acutely rapamycin-sensitive mTOR complex 1 (mTORC1) is defined by a core subunit Raptor, whereas mTORC2 lacks Raptor and, instead, has Rictor and SIN1 as distinct essential components. We now show that homeostasis and function of B cells require Rictor. Conditional deletion of Rictor before lymphoid specification impaired generation of mature follicular, marginal zone, and B1a B lymphocytes. Induced inactivation in adult mice caused cell-autonomous defects in B lymphoid homeostasis and antibody responses in vivo, along with affecting plasma cells in bone marrow. Survival of B lymphocytes depended on Rictor, which was vital for normal induction of prosurvival genes, suppression of proapoptotic genes, nuclear factor κB induction after B-cell receptor stimulation, and B-cell activating factor-induced nuclear factor κB2/p52 generation. Collectively, the findings provide evidence that mTOR signaling affects survival and proliferation of mature B lymphocytes, and establish Rictor as an important signal relay in B-cell homeostasis, fate, and functions.
Type 1 cannabinoid receptor blockers increase high-density lipoprotein cholesterol levels. Although genetic variation in the type 1 cannabinoid receptor--encoded by the CNR1 gene--is known to influence high-density lipoprotein cholesterol level as well, human studies conducted to date have been limited to genetic markers such as haplotype-tagging single nucleotide polymorphisms. Here we identify rs806371 in the CNR1 promoter as the causal variant. We re-sequence the CNR1 gene and genotype all variants in a DNA biobank linked to comprehensive electronic medical records. By testing each variant for association with high-density lipoprotein cholesterol level in a clinical practice-based setting, we localize a putative functional allele to a 100-bp window in the 5'-flanking region. Assessment of variants in this window for functional impact on electrophoretic mobility shift assay identifies rs806371 as a novel regulatory binding element. Reporter gene assays confirm that rs806371 reduces gene expression, thereby linking CNR1 gene variation to high-density lipoprotein cholesterol level in humans.
Analysis of 4,405 variants in 89,050 European subjects from 41 case-control studies identified three independent association signals for estrogen-receptor-positive tumors at 11q13. The strongest signal maps to a transcriptional enhancer element in which the G allele of the best candidate causative variant rs554219 increases risk of breast cancer, reduces both binding of ELK4 transcription factor and luciferase activity in reporter assays, and may be associated with low cyclin D1 protein levels in tumors. Another candidate variant, rs78540526, lies in the same enhancer element. Risk association signal 2, rs75915166, creates a GATA3 binding site within a silencer element. Chromatin conformation studies demonstrate that these enhancer and silencer elements interact with each other and with their likely target gene, CCND1.
Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.
Archaeal and eukaryotic B-family DNA polymerases (pols) mainly replicate chromosomal DNA but stall at lesions, which are often bypassed with Y-family pols. In this study, a B-family pol Vent (exo(-)) from the euryarchaeon Thermococcus litoralis was studied with three types of DNA lesions-N(2)-alkylG, O(6)-alkylG, and an abasic (AP) site-in comparison with a model Y-family pol Dpo4 from Sulfolobus solfataricus, to better understand the effects of various DNA modifications on binding, bypass efficiency, and fidelity of pols. Vent (exo(-)) readily bypassed N(2)-methyl(Me)G and O(6)-MeG, but was strongly blocked at O(6)-benzyl(Bz)G and N(2)-BzG, whereas Dpo4 efficiently bypassed N(2)-MeG and N(2)-BzG and partially bypassed O(6)-MeG and O(6)-BzG. Vent (exo(-)) bypassed an AP site to an extent greater than Dpo4, corresponding with steady-state kinetic data. Vent (exo(-)) showed ~110-, 180-, and 300-fold decreases in catalytic efficiency (k(cat)/K(m)) for nucleotide insertion opposite an AP site, N(2)-MeG, and O(6)-MeG but ~1800- and 5000-fold decreases opposite O(6)-BzG and N(2)-BzG, respectively, as compared to G, whereas Dpo4 showed little or only ~13-fold decreases opposite N(2)-MeG and N(2)-BzG but ~260-370-fold decreases opposite O(6)-MeG, O(6)-BzG, and the AP site. Vent (exo(-)) preferentially misinserted G opposite N(2)-MeG, T opposite O(6)-MeG, and A opposite an AP site and N(2)-BzG, while Dpo4 favored correct C insertion opposite those lesions. Vent (exo(-)) and Dpo4 both bound modified DNAs with affinities similar to unmodified DNA. Our results indicate that Vent (exo(-)) is as or more efficient as Dpo4 in synthesis opposite O(6)-MeG and AP lesions, whereas Dpo4 is much or more efficient opposite (only) N(2)-alkylGs than Vent (exo(-)), irrespective of DNA-binding affinity. Our data also suggest that Vent (exo(-)) accepts nonbulky DNA lesions (e.g., N(2)- or O(6)-MeG and an AP site) as manageable substrates despite causing error-prone synthesis, whereas Dpo4 strongly favors minor-groove N(2)-alkylG lesions over major-groove or noninstructive lesions.