Exploration of intramolecular split G-quadruplex and its analytical applications.

Distinct from intermolecular split G-quadruplex (Inter-SG), intramolecular split G-quadruplex (Intra-SG) which could be generated in a DNA spacer-inserted G-quadruplex strand has not been systematically explored. Not only is it essential for the purpose of simplicity of DNA-based bioanalytical applications, but also it will give us hints how to design split G-quadruplex-based system. Herein, comprehensive information is provided about influences of spacer length and split mode on the formation of Intra-SG, how to adjust its thermodynamic stability, and selection of optimal Intra-SG for bioanalysis. For instances, non-classical Intra-SG (e.g. 2:10, 4:8 and 5:7) displays lower stability than classical split strands (3:9, 6:6 and 9:3), which is closely related to integrity of consecutive guanine tract; as compared to regular Intra-SG structures, single-thymine capped ones have reduced melting temperature, providing an effective approach to adjustment of stability. It is believed that the disclosed rules in this study will contribute to the effective application of split G-quadruplex in the field of DNA technology in the future.

Probing the Behaviour of Cas1-Cas2 upon Protospacer Binding in CRISPR-Cas Systems using Molecular Dynamics Simulations.

Adaptation in CRISPR-Cas systems enables the generation of an immunological memory to defend against invading viruses. This process is driven by foreign DNA spacer (termed protospacer) selection and integration mediated by Cas1-Cas2 protein. Recently, different states of Cas1-Cas2, in its free form and in complex with protospacer DNAs, were solved by X-ray crystallography. In this paper, molecular dynamics (MD) simulations are employed to study crystal structures of one free and two protospacer-bound Cas1-Cas2 complexes. The simulated results indicate that the protospacer binding markedly increases the system stability, in particular when the protospacer containing the PAM-complementary sequence. The hydrogen bond and binding free energy calculations explain that PAM recognition introduces more specific interactions to increase the cleavage activity of Cas1. By using principal component analysis (PCA) and intramolecular angle calculation, this study observes two dominant slow motions associated with the binding of Ca1-Cas2 to the protospacer and potential target DNAs respectively. The comparison of DNA structural deformation further implies a cooperative conformational change of Cas1-Cas2 and protospacer for the target DNA capture. We propose that this cooperativity is the intrinsic requirement of the CRISPR integration complex formation. This study provides some new insights into the understanding of CRISPR-Cas adaptation.

Mutation in a flexible linker modulates binding affinity for modular complexes.

Tandem beta zippers are modular complexes formed between repeated linear motifs and tandemly arrayed domains of partner proteins in which ß-strands form upon binding. Studies of such complexes, formed by LIM domain proteins and linear motifs in their intrinsically disordered partners, revealed spacer regions between the linear motifs that are relatively flexible but may affect the overall orientation of the binding modules. We demonstrate that mutation of a solvent exposed side chain in the spacer region of an LHX4-ISL2 complex has no significant effect on the structure of the complex, but decreases binding affinity, apparently by increasing flexibility of the linker.

A tiered hidden Markov model characterizes multi-scale chromatin states.

Precise characterization of chromatin states is an important but difficult task for understanding the regulatory role of chromatin. A number of computational methods have been developed with varying levels of success. However, a remaining challenge is to model epigenomic patterns over multi-scales, as each histone mark is distributed with its own characteristic length scale. We developed a tiered hidden Markov model and applied it to analyze a ChIP-seq dataset in human embryonic stem cells. We identified a two-tier structure containing 15 distinct bin-level chromatin states grouped into three domain-level states. Whereas the bin-level states capture the local variation of histone marks, the domain-level states detect large-scale variations. Compared to bin-level states, the domain-level states are more robust and coherent. We also found active regions in intergenic regions that upon closer examination were expressed non-coding RNAs and pseudogenes. These results provide insights into an additional layer of complexity in chromatin organization.

Organization of 5S rDNA in species of the fish Leporinus: two different genomic locations are characterized by distinct nontranscribed spacers.

To address understanding the organization of the 5S rRNA multigene family in the fish genome, the nucleotide sequence and organization array of 5S rDNA were investigated in the genus Leporinus, a representative freshwater fish group of South American fauna. PCR, subgenomic library screening, genomic blotting, fluorescence in situ hybridization, and DNA sequencing were employed in this study. Two arrays of 5S rDNA were identified for all species investigated, one consisting of monomeric repeat units of around 200 bp and another one with monomers of 900 bp. These 5S rDNA arrays were characterized by distinct NTS sequences (designated NTS-I and NTS-II for the 200- and 900-bp monomers, respectively); however, their coding sequences were nearly identical. The 5S rRNA genes were clustered in two chromosome loci, a major one corresponding to the NTS-I sites and a minor one corresponding to the NTS-II sites. The NTS-I sequence was variable among Leporinus spp., whereas the NTS-II was conserved among them and even in the related genus Schizodon. The distinct 5S rDNA arrays might characterize two 5S rRNA gene subfamilies that have been evolving independently in the genome.

Bent DNA in the large intergenic region of wheat dwarf geminivirus.

Wheat dwarf virus (WDV) is a member of the geminivirus group, unique plant DNA viruses which replicate exclusively via dsDNA replication intermediates. The large intergenic region (LIR), a nontranscribed regulatory region, contains an inverted repeat with the potential to form a stem-loop structure in which the initiation site for WDV (+)strand (virion-sense) DNA replication has been mapped. In this work, we have studied by two-dimensional electrophoresis and by electron microscopy the DNA structure of the WDV LIR and we have identified a DNA sequence which confers a static DNA curvature. This is the first report of the occurrence of DNA bending in the regulatory region of a geminivirus genome. The bending locus, defined by an 80-bp cluster of A(T) tracts, is located downstream from the potential stem-loop. The A(T) tract proximal to the stem-loop structure is separated by 14 bp from the inverted repeat, and deletion of distal A(T) tracts abolished the curvature of the new WDV deltaLIR. The bending center maps approximately 80 bp downstream from the putative initiation site for (+)strand DNA replication and approximately 70 bp upstream from the TATA box for virion-sense transcription. The possible implications of the DNA bending locus as a regulatory element of WDV DNA replication and/or virion-sense transcription are discussed.