Structure of two intramolecular G-quadruplexes formed by natural human telomere sequences in K+ solution

Intramolecular G-quadruplexes formed by human telomere sequences are attractive anticancer targets. Recently, four-repeat human telomere sequences have been shown to form two different intramolecular (3 + 1) G-quadruplexes in K(+) solution (Form 1 and Form 2). Here we report on the solution structures of both Form 1 and Form 2 adopted by natural human telomere sequences. Both structures contain the (3 + 1) G-tetrad core with one double-chain-reversal and two edgewise loops, but differ in the successive order of loop arrangements within the G-quadruplex scaffold. Our results provide the structural details at the two ends of the G-tetrad core in the context of natural sequences and information on different loop conformations. This structural information might be important for our understanding of telomere G-quadruplex structures and for anticancer drug design targeted to such scaffolds.     

Scl binds to primed enhancers in mesoderm to regulate hematopoietic and cardiac fate divergence

Scl/Tal1 confers hemogenic competence and prevents ectopic cardiomyogenesis in embryonic endothelium by unknown mechanisms. We discovered that Scl binds to hematopoietic and cardiac enhancers that become epigenetically primed in multipotent cardiovascular mesoderm, to regulate the divergence of hematopoietic and cardiac lineages. Scl does not act as a pioneer factor but rather exploits a pre‐established epigenetic landscape. As the blood lineage emerges, Scl binding and active epigenetic modifications are sustained in hematopoietic enhancers, whereas cardiac enhancers are decommissioned by removal of active epigenetic marks. Our data suggest that, rather than recruiting corepressors to enhancers, Scl prevents ectopic cardiogenesis by occupying enhancers that cardiac factors, such as Gata4 and Hand1, use for gene activation. Although hematopoietic Gata factors bind with Scl to both activated and repressed genes, they are dispensable for cardiac repression, but necessary for activating genes that enable hematopoietic stem/progenitor cell development. These results suggest that a unique subset of enhancers in lineage‐specific genes that are accessible for regulators of opposing fates during the time of the fate decision provide a platform where the divergence of mutually exclusive fates is orchestrated.     

Tissue- and development-specific distributions of cytoskeletal elements in growing cells of the maize root apex

ABSTRACT

Indirect immunofluorescence performed using sections of actively growing maize root apices fixed and then embedded in low-melting-point Steedman's wax has proved efficient in revealing the arrangements and reorganizations of motility-related cytoskeletal elements which are associated with root cell development and tissue differentiation. This powerful, yet relatively simple, technique shows that specific rearrangements of both microtubular (MT) and actin microfilament (MF) arrays occur in cells as they leave the meristem and traverse the transitional region interpolated between meristem and elongation region. Cytoskeletal and growth analyses have identified the transition zone as critical for both cell and root development; it is in this zone that cell growth is channelled, by the cytoskeleton, into a strictly polarized mode which enables root tips to extend rapidly through the soil in search of water and nutrients. An integrated cytoskeletal network is crucial for both the cytomorphogenesis of individual cells and the overall morphogenesis of the plant body. The latter process can be viewed as a reflection of the tight control which cytoskeletal networks exert not only over cell division planes in the cells within meristematic apices but also over the orientation of cell growth in the meristem and elsewhere. Endoplasmic MTs interconnecting the plasma membrane with the nucleus are suggested to be involved in cell division control; they may also act as a two-way cytoskeletal communication channel for signals passing to and fro between the extracellular environment and the genome. Moreover, the dynamism of endoplasmic MTs exerts direct effects on chromatin structure and the accompanying nuclear architecture and hence can help exert a cellular level of control over cell growth and cell cycle progression. Because the inherent dynamic instability of MTs depends on the concentration of tubulin dimers within the cytoplasm, we propose that when asymmetric cell division occurs, it will result in two daughter cells which differ in the turnover rates of their MTs. This phenomenon could be responsible for different cell fates of daughter plant cells produced by such cell divisions.     

DNAJC9 integrates heat shock molecular chaperones into the histone chaperone network

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Human GLTP: Three distinct functions for the three tryptophans in a novel peripheral amphitropic fold

Human glycolipid transfer protein (GLTP) serves as the GLTP-fold prototype, a novel, to our knowledge, peripheral amphitropic fold and structurally unique lipid binding motif that defines the GLTP superfamily. Despite conservation of all three intrinsic Trps in vertebrate GLTPs, the Trp functional role(s) remains unclear. Herein, the issue is addressed using circular dichroism and fluorescence spectroscopy along with an atypical Trp point mutation strategy. Far-ultraviolet and near-ultraviolet circular dichroism spectroscopic analyses showed that W96F-W142Y-GLTP and W96Y-GLTP retain their native conformation and stability, whereas W85Y-W96F-GLTP is slightly altered, in agreement with relative glycolipid transfer activities of >90%, ∼85%, and ∼45%, respectively. In silico three-dimensional modeling and acrylamide quenching of Trp fluorescence supported a nativelike folding conformation. With the Trp⁹⁶-less mutants, changes in emission intensity, wavelength maximum, lifetime, and time-resolved anisotropy decay induced by phosphoglyceride membranes lacking or containing glycolipid and by excitation at different wavelengths along the absorption-spectrum red edge indicated differing functions for W142 and W85. The data suggest that W142 acts as a shallow-penetration anchor during docking with membrane interfaces, whereas the buried W85 indole helps maintain proper folding and possibly regulates membrane-induced transitioning to a glycolipid-acquiring conformation. The findings illustrate remarkable versatility for Trp, providing three distinct intramolecular functions in the novel amphitropic GLTP fold.     

Recognition of a mononucleosomal histone modification pattern by BPTF via multivalent interactions

Little is known about how combinations of histone marks are interpreted at the level of nucleosomes. The second PHD finger of human BPTF is known to specifically recognize histone H3 when methylated on lysine 4 (H3K4me2/3). Here, we examine how additional heterotypic modifications influence BPTF binding. Using peptide surrogates, three acetyllysine ligands are indentified for a PHD-adjacent bromodomain in BPTF via systematic screening and biophysical characterization. Although the bromodomain displays limited discrimination among the three possible acetyllysines at the peptide level, marked selectivity is observed for only one of these sites, H4K16ac, in combination with H3K4me3 at the mononucleosome level. In support, these two histone marks constitute a unique trans-histone modification pattern that unambiguously resides within a single nucleosomal unit in human cells, and this module colocalizes with these marks in the genome. Together, our data call attention to nucleosomal patterning of covalent marks in dictating critical chromatin associations.     

Structural basis for self-cleavage prevention by tag:anti-tag pairing complementarity in type VI Cas13 CRISPR systems

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Implications for damage recognition during Dpo4-mediated mutagenic bypass of m1G and m3C lesions

DNA is susceptible to alkylation damage by a number of environmental agents that modify the Watson-Crick edge of the bases. Such lesions, if not repaired, may be bypassed by Y-family DNA polymerases. The bypass polymerase Dpo4 is strongly inhibited by 1-methylguanine (m1G) and 3-methylcytosine (m3C), with nucleotide incorporation opposite these lesions being predominantly mutagenic. Further, extension after insertion of both correct and incorrect bases, introduces additional base substitution and deletion errors. Crystal structures of the Dpo4 ternary extension complexes with correct and mismatched 3'-terminal primer bases opposite the lesions reveal that both m1G and m3C remain positioned within the DNA template/primer helix. However, both correct and incorrect pairing partners exhibit pronounced primer terminal nucleotide distortion, being primarily evicted from the DNA helix when opposite m1G or misaligned when pairing with m3C. Our studies provide insights into mechanisms related to hindered and mutagenic bypass of methylated lesions and models associated with damage recognition by repair demethylases.     

Structure–activity relationships at a nucleobase-stacking tryptophan required for chemomechanical coupling in the DNA resecting motor-nuclease AdnAB

Mycobacterial AdnAB is a heterodimeric helicase-nuclease that initiates homologous recombination by resecting DNA double-strand breaks. The AdnB subunit hydrolyzes ATP to drive single-nucleotide steps of 3′-to-5′ translocation of AdnAB on the tracking DNA strand via a ratchet-like mechanism. Trp325 in AdnB motif III, which intercalates into the tracking strand and makes a π stack on a nucleobase 5′ of a flipped-out nucleoside, is the putative ratchet pawl without which ATP hydrolysis is mechanically futile. Here, we report that AdnAB mutants wherein Trp325 was replaced with phenylalanine, tyrosine, histidine, leucine, or alanine retained activity in ssDNA-dependent ATP hydrolysis but displayed a gradient of effects on DSB resection. The resection velocities of Phe325 and Tyr325 mutants were 90% and 85% of the wild-type AdnAB velocity. His325 slowed resection rate to 3% of wild-type and Leu325 and Ala325 abolished DNA resection. A cryo-EM structure of the DNA-bound Ala325 mutant revealed that the AdnB motif III peptide was disordered and the erstwhile flipped out tracking strand nucleobase reverted to a continuous base-stacked arrangement with its neighbors. We conclude that π stacking of Trp325 on a DNA nucleobase triggers and stabilizes the flipped-out conformation of the neighboring nucleoside that underlies formation of a ratchet pawl.