Molecular Mechanism of Methyl-Dependent and Spatial-Specific DNA Recognition of c-Jun Homodimere

DNA methylation is important in regulation of gene expression and normal development because it alters the interplay between protein and DNA. Experiments have shown that a single 5-methylcytosine at different CpG sites (mCpG) might have different effects on specific recognition, but the atomistic origin and dynamic details are largely unclear. In this work, we investigated the mechanism of monomethylation at different CpG sites in the cognate motif and the cooperativity of full methylation. By constructing four models of c-Jun/Jun protein binding to the 5’-XGAGTCA -3’(X represents C or methylated C) motif, we characterized the dynamics of the contact interface using the all-atom molecular dynamics method. Free energy analysis of MM/GBSA suggests that regardless of whether the C12pG13 site of the bottom strand is methylated, the effects from mC25pG26 of the top strand are dominant and can moderately enhance the binding by ~ 31kcal/mol, whereas mC12pG13 showed a relatively small contribution, in agreement with the experimental data. Remarkably, we found that this spatial-specific influence was induced by different regulatory rules. The influence of the mC25pG26 site is mainly mediated by steric hindrance. The additional methyl group leads to the conformational changes in nearby residues and triggers an obvious structural bending in the protein, which results in the formation of a new T-ASN-C triad that enhances the specific recognition of TCA half-sites. The substitution of the methyl group at the C12pG13 site of the bottom strand breaks the original H-bonds directly. Such changes in electrostatic interactions also lead to the remote allosteric effects of protein by multifaceted interactions but have negligible contributions to binding. Although these two influence modes are different, they can both fine-tune the local environment, which might produce remote allosteric effects through protein-protein interactions. Further analysis reveals that the discrepancies in these two modes are primarily due to their location. Moreover, when both sites are methylated, the major determinant of binding specificity depends on the context and the location of the methylation site, which is the result of crosstalk and cooperativity.

RIG-I ATPase Activity and Discrimination of Self-RNA versus Non-Self-RNA

ABSTRACTMany RNA viruses are detected by retinoic acid-inducible gene i (RIG-I), a cytoplasmic sensor that triggers an antiviral response upon binding non-self-RNA that contains a stretch of double-stranded RNA (dsRNA) bearing a base-paired 5′ ppp nucleotide. To gain insight into how RIG-I discriminates between self-RNA and non-self-RNA, we used duplexes whose complementary bottom strand contained both ribo- and deoxynucleotides. These duplexes were examined for their binding to RIG-I and their relative abilities to stimulate ATPase activity, to induce RIG-I dimerization on the duplex, and to induce beta interferon (IFN-β) expression. We show that the chemical nature of the bottom strand is not critical for RIG-I binding. However, two key ribonucleotides, at positions 2 and 5 on the bottom strand, are minimally required for the RIG-I ATPase activity, which is necessary but not sufficient for IFN-β stimulation. We find that duplexes with shorter stretches of dsRNA, as model self-RNAs, bind less stably to RIG-I but nevertheless have an enhanced ability to stimulate the ATPase. Moreover, ATPase activity promotes RIG-I recycling on RIG-I/dsRNA complexes. Since pseudo-self-RNAs bind to RIG-I less stably, they are preferentially recycled by ATP hydrolysis that weakens the helicase domain binding of dsRNA. Our results suggest that one function of the ATPase is to restrict RIG-I signaling to its interaction with non-self-RNA. A model of how this discrimination occurs as a function of dsRNA length is presented.IMPORTANCEThe innate immune response to pathogens is based on the discrimination between self-RNA and non-self-RNA. The main determinants of this detection for RNA viruses are specific pathogen-associated molecular patterns (PAMPs) of RNA, which are detected by dedicated cytoplasmic pattern recognition receptors (PRRs). RIG-I is a PRR that specifically detects short viral dsRNAs amid a sea of cellular RNAs. Here we study the determinants of this discrimination and how RIG-I ATPase activity, the only enzymatic activity of this sensor, contributes to its activation in a manner restricted to its interaction with non-self-RNAs. We also show how the innate immune response evolves during infection via IFN expression, from a state in which discrimination of self-RNA from non-self-RNA is most important to one in which this discrimination is sacrificed for the effectiveness of the antiviral response.

An Entamoeba histolytica LINE/SINE Pair Inserts at Common Target Sites Cleaved by the Restriction Enzyme-Like LINE-Encoded Endonuclease

ABSTRACT The non-long-terminal-repeat (non-LTR) retrotransposons (also called long interspersed repetitive elements [LINEs]) are among the oldest retroelements. Here we describe the properties of such an element from a primitive protozoan parasite, Entamoeba histolytica, that infects the human gut. This 4.8-kb element, called EhLINE1, is present in about 140 copies dispersed throughout the genome. The element belongs to the R4 clade of non-LTR elements. It has a centrally located reverse transcriptase domain and a restriction enzyme-like endonuclease (EN) domain at the carboxy terminus. We have cloned and expressed a 794-bp fragment containing the EN domain in Escherichia coli. The purified protein could nick supercoiled pBluescript DNA to yield open circular and linear DNAs. The conserved PDX12-14D motif was required for activity. Genomic sequences flanking the sites of insertion of EhLINE1 and the putative partner short interspersed repetitive element (SINE), EhSINE1, were analyzed. Both elements resulted in short target site duplications (TSD) upon insertion. A common feature was the presence of a short T-rich stretch just upstream of the TSD in most insertion sites. By sequence analysis an empty target site in the E. histolytica genome, known to be occupied by EhSINE1, was identified. When a 176-bp fragment containing the empty site was used as a substrate for EN, it was prominently nicked on the bottom strand at the precise point of insertion of EhSINE1, showing that this SINE could use the LINE-encoded endonuclease for its insertion. The nick on the bottom strand was toward the right of the TSD, which is uncommon. The lack of strict target site-specificity of the restriction enzyme-like EN encoded by EhLINE1 is also exceptional. A model for retrotransposition of EhLINE1/SINE1 is presented.

Defining cosQ, the Site Required for Termination of Bacteriophage λ DNA Packaging

Bacteriophage λ is a double-stranded DNA virus that processes concatemeric DNA into virion chromosomes by cutting at specific recognition sites termed cos. A cos is composed of three subsites: cosN, the nicking site; cosB, required for packaging initiation; and cosQ, required for termination of chromosome packaging. During packaging termination, nicking of the bottom strand of cosN depends on cosQ, suggesting that cosQ is needed to deliver terminase to the bottom strand of cosN to carry out nicking. In the present work, saturation mutagenesis showed that a 7-bp segment comprises cosQ. A proposal that cosQ function requires an optimal sequence match between cosQ and cosNR, the right cosN half-site, was tested by constructing double cosQ mutants; the behavior of the double mutants was inconsistent with the proposal. Substitutions in the 17-bp region between cosQ and cosN resulted in no major defects in chromosome packaging. Insertional mutagenesis indicated that proper spacing between cosQ and cosN is required. The lethality of integral helical insertions eliminated a model in which DNA looping enables cosQ to deliver a gpA protomer for nicking at cosN. The 7 bp of cosQ coincide exactly with the recognition sequence for the Escherichia coli restriction endonuclease, EcoO109I.

Sequence-Specific Recognition and Cleavage of Telomeric Repeat (TTAGG)n by Endonuclease of Non-Long Terminal Repeat Retrotransposon TRAS1

ABSTRACT The telomere of the silkworm Bombyx mori consists of (TTAGG/CCTAA) n repeats and harbors a large number of telomeric repeat-specific non-long terminal repeat retrotransposons, such as TRAS1 and SART1. To understand how these retrotransposons recognize and integrate into the telomeric repeat in a sequence-specific manner, we expressed the apurinic-apryrimidinic endonuclease-like endonuclease domain of TRAS1 (TRAS1 EN), which is supposed to digest the target DNA, and characterized its enzymatic properties. Purified TRAS1 EN could generate specific nicks on both strands of the telomeric repeat sequence between T and A of the (TTAGG) n strand (bottom strand) and between C and T of the (CCTAA) n strand (top strand). These sites are consistent with insertion sites expected from the genomic structure of boundary regions of TRAS1. Time course studies of nicking activities on both strands revealed that the cleavages on the bottom strand preceded those on the top strand, supporting the target-primed reverse transcription model. TRAS1 EN could cleave the telomeric repeats specifically even if it was flanked by longer tracts of nontelomeric sequence, indicating that the target site specificity of the TRAS1 element was mainly determined by its EN domain. Based on mutation analyses, TRAS1 EN recognizes less than 10 bp around the initial cleavage site (upstream 7 bp and downstream 3 bp), and the GTTAG sequence especially is essential for the cleavage reaction on the bottom strand (5′. . . TTAGGTT ↓ AGG . . . 3′). TRAS1 EN, the first identified endonuclease digesting telomeric repeats, may be used as a genetic tool to shorten the telomere in insects and some other organisms.

Footprint Analysis of the RAG Protein Recombination Signal Sequence Complex for V(D)J Type Recombination

ABSTRACT We have studied the interaction between recombination signal sequences (RSSs) and protein products of the truncated forms of recombination-activating genes (RAG) by gel mobility shift, DNase I footprinting, and methylation interference assays. Methylation interference with dimethyl sulfate demonstrated that binding was blocked by methylation in the nonamer at the second-position G residue in the bottom strand and at the sixth- and seventh-position A residues in the top strand. DNase I footprinting experiments demonstrated that RAG1 alone, or even a RAG1 homeodomain peptide, gave footprint patterns very similar to those obtained with the RAG1-RAG2 complex. In the heptamer, partial methylation interference was observed at the sixth-position A residue in the bottom strand. In DNase I footprinting, the heptamer region was weakly protected in the bottom strand by RAG1. The effects of RSS mutations on RAG binding were evaluated by DNA footprinting. Comparison of the RAG-RSS footprint data with the published Hin model confirmed the notion that sequence-specific RSS-RAG interaction takes place primarily between the Hin domain of the RAG1 protein and adjacent major and minor grooves of the nonamer DNA.