NAR Breakthrough Article: A dimeric state for PRC2

Polycomb repressive complex-2 (PRC2) is a histone methyltransferase required for epigenetic silencing during development and cancer. Long non-coding RNAs (lncRNAs) can recruit PRC2 to chromatin. Previous studies identified PRC2 subunits in a complex with the apparent molecular weight of a dimer, which might be accounted for by the incorporation of additional protein subunits or RNA rather than PRC2 dimerization. Here we show that reconstituted human PRC2 is in fact a dimer, using multiple independent approaches including analytical size exclusion chromatography (SEC), SEC combined with multi-angle light scattering and co-immunoprecipitation of differentially tagged subunits. Even though it contains at least two RNA-binding subunits, each PRC2 dimer binds only one RNA molecule. Yet, multiple PRC2 dimers bind a single RNA molecule cooperatively. These observations suggest a model in which the first RNA binding event promotes the recruitment of multiple PRC2 complexes to chromatin, thereby nucleating repression.     

NAR Breakthrough Article: Pre-activation of the genome integrity checkpoint increases DNA damage tolerance

The genome integrity checkpoint is a conserved signaling pathway that is regulated in yeast by the Mec1 (homologous to human ATR) and Rad53 (homologous to human Chk1) kinases. The pathway coordinates a multifaceted response that allows cells to cope with DNA damage and DNA replication stress. The full activation of the checkpoint blocks origin firing, stabilizes replication forks, activates DNA repair proteins and may lead to senescence or apoptosisin higher eukaryotes. We have recently demonstrated that endogenous replication stress can activate the genome integrity checkpoint in budding yeast at a low level that does not go so far as to interfere with cell cycle progression, but it does activate DNA damage-inducible proteins. Here we demonstrate that the low level pre-activation of the checkpoint, either by endogenous replication stress or by the nucleotide-depleting drug hydroxyurea, can increase damage tolerance to multiple DNA-damaging agents. These results may provide new strategies for using the checkpoint to protect normal cells from genotoxic stress.     

NAR Breakthrough Article: Structural basis of lariat RNA recognition by the intron debranching enzyme Dbr1

The enzymatic processing of cellular RNA molecules requires selective recognition of unique chemical and topological features. The unusual 2′,5′-phosphodiester linkages in RNA lariats produced by the spliceosome must be hydrolyzed by the intron debranching enzyme (Dbr1) before they can be metabolized or processed into essential cellular factors, such as snoRNA and miRNA. Dbr1 is also involved in the propagation of retrotransposons and retroviruses, although the precise role played by the enzyme in these processes is poorly understood. Here, we report the first structures of Dbr1 alone and in complex with several synthetic RNA compounds that mimic the branchpoint in lariat RNA. The structures, together with functional data on Dbr1 variants, reveal the molecular basis for 2′,5′-phosphodiester recognition and explain why the enzyme lacks activity toward 3′,5′-phosphodiester linkages. The findings illuminate structure/function relationships in a unique enzyme that is central to eukaryotic RNA metabolism and set the stage for the rational design of inhibitors that may represent novel therapeutic agents to treat retroviral infections and neurodegenerative disease.     

NAR Breakthrough Article: FDF-PAGE: a powerful technique revealing previously undetected small RNAs sequestered by complementary transcripts

Small RNAs, between 18nt and 30nt in length, are a diverse class of non-coding RNAs that mediate a range of cellular processes, from gene regulation to pathogen defense. They guide ribonucleoprotein complexes to their target nucleic acids by Watson–Crick base pairing. We report here that current techniques for small RNA detection and library generation are biased by formation of RNA duplexes. To address this problem, we established FDF-PAGE (fully-denaturing formaldehyde polyacrylamide gel electrophoresis) to prevent annealing of sRNAs to their complement. By applying FDF-PAGE, we provide evidence that both strands of viral small RNA are present in near equimolar ratios, indicating that the predominant precursor is a long double-stranded RNA. Comparing non-denaturing conditions to FDF-PAGE uncovered extensive sequestration of miRNAs in model organisms and allowed us to identify candidate small RNAs under the control of competing endogenous RNAs (ceRNAs). By revealing the full repertoire of small RNAs, we can begin to create a better understanding of small RNA mediated interactions.     

Sequence-specific cleavage of RNA using chimeric DNA splints and RNase H

To cleave RNA molecules using E. coli RNase H in a site-specific manner, a short oligodeoxyribonucleotide (3-5 mer) linked with oligo(2'-O-methyl)ribonucleotide(s) was designed to be used as a DNA splint. Our model experiments with ribooligomer the splint duplexes (9 mers) and RNase H demonstrated that a tetradeoxynucleotide cluster seems to be sufficient for the enzyme recognition and the short DNA-containing splint directs a unique cleavage of RNA by RNase H. The method could be applied to longer ribooligonucleotide substrates. For example, when 3'm (GA)d(AGAA)m(GGU)5' was used as a hybridization strand, 32pUCUUUCUUCUUCCAGGAU was cleaved specifically between U11 and C12 to yield 32pUCUUUCUUCUU. This method will have a variety of applications for the study of RNA.     

NAR Breakthrough Article: Helq acts in parallel to Fancc to suppress replication-associated genome instability

HELQ is a superfamily 2 DNA helicase found in archaea and metazoans. It has been implicated in processing stalled replication forks and in repairing DNA double-strand breaks and inter-strand crosslinks. Though previous studies have suggested the possibility that HELQ is involved in the Fanconi anemia (FA) pathway, a dominant mechanism for inter-strand crosslink repair in vertebrates, this connection remains elusive. Here, we investigated this question in mice using the Helq^gt and Fancc⁻ strains. Compared with Fancc^−/− mice lacking FANCC, a component of the FA core complex, Helq^gt/gt mice exhibited a mild of form of FA-like phenotypes including hypogonadism and cellular sensitivity to the crosslinker mitomycin C. However, unlike Fancc^−/− primary fibroblasts, Helq^gt/gt cells had intact FANCD2 mono-ubiquitination and focus formation. Notably, for all traits examined, Helq was non-epistatic with Fancc, as Helq^gt/gt;Fancc^−/− double mutants displayed significantly worsened phenotypes than either single mutant. Importantly, this was most noticeable for the suppression of spontaneous chromosome instability such as micronuclei and 53BP1 nuclear bodies, known consequences of persistently stalled replication forks. These findings suggest that mammalian HELQ contributes to genome stability in unchallenged conditions through a mechanism distinct from the function of FANCC.     

NAR Breakthrough Article: Highlights of the DNA cutters: a short history of the restriction enzymes

In the early 1950’s, ‘host-controlled variation in bacterial viruses’ was reported as a non-hereditary phenomenon: one cycle of viral growth on certain bacterial hosts affected the ability of progeny virus to grow on other hosts by either restricting or enlarging their host range. Unlike mutation, this change was reversible, and one cycle of growth in the previous host returned the virus to its original form. These simple observations heralded the discovery of the endonuclease and methyltransferase activities of what are now termed Type I, II, III and IV DNA restriction-modification systems. The Type II restriction enzymes (e.g. EcoRI) gave rise to recombinant DNA technology that has transformed molecular biology and medicine. This review traces the discovery of restriction enzymes and their continuing impact on molecular biology and medicine.     

NAR Breakthrough Article: A systematic survey of the Cys2His2 zinc finger DNA-binding landscape

Cys₂His₂ zinc fingers (C2H2-ZFs) comprise the largest class of metazoan DNA-binding domains. Despite this domain''s well-defined DNA-recognition interface, and its successful use in the design of chimeric proteins capable of targeting genomic regions of interest, much remains unknown about its DNA-binding landscape. To help bridge this gap in fundamental knowledge and to provide a resource for design-oriented applications, we screened large synthetic protein libraries to select binding C2H2-ZF domains for each possible three base pair target. The resulting data consist of >160 000 unique domain–DNA interactions and comprise the most comprehensive investigation of C2H2-ZF DNA-binding interactions to date. An integrated analysis of these independent screens yielded DNA-binding profiles for tens of thousands of domains and led to the successful design and prediction of C2H2-ZF DNA-binding specificities. Computational analyses uncovered important aspects of C2H2-ZF domain–DNA interactions, including the roles of within-finger context and domain position on base recognition. We observed the existence of numerous distinct binding strategies for each possible three base pair target and an apparent balance between affinity and specificity of binding. In sum, our comprehensive data help elucidate the complex binding landscape of C2H2-ZF domains and provide a foundation for efforts to determine, predict and engineer their DNA-binding specificities.     

NAR Breakthrough Article: Next-generation sequencing reveals the biological significance of the N2,3-ethenoguanine lesion in vivo

Etheno DNA adducts are a prevalent type of DNA damage caused by vinyl chloride (VC) exposure and oxidative stress. Etheno adducts are mutagenic and may contribute to the initiation of several pathologies; thus, elucidating the pathways by which they induce cellular transformation is critical. Although N²,3-ethenoguanine (N²,3-εG) is the most abundant etheno adduct, its biological consequences have not been well characterized in cells due to its labile glycosidic bond. Here, a stabilized 2′-fluoro-2′-deoxyribose analog of N²,3-εG was used to quantify directly its genotoxicity and mutagenicity. A multiplex method involving next-generation sequencing enabled a large-scale in vivo analysis, in which both N²,3-εG and its isomer 1,N²-ethenoguanine (1,N²-εG) were evaluated in various repair and replication backgrounds. We found that N²,3-εG potently induces G to A transitions, the same mutation previously observed in VC-associated tumors. By contrast, 1,N²-εG induces various substitutions and frameshifts. We also found that N²,3-εG is the only etheno lesion that cannot be repaired by AlkB, which partially explains its persistence. Both εG lesions are strong replication blocks and DinB, a translesion polymerase, facilitates the mutagenic bypass of both lesions. Collectively, our results indicate that N²,3-εG is a biologically important lesion and may have a functional role in VC-induced or inflammation-driven carcinogenesis.     

Sequence-specific RNase H cleavage of gag mRNA from HIV-1 infected cells by an antisense oligonucleotide in vitro.

We have used a ribonuclease protection assay to investigate RNase H cleavage of HIV-1 mRNA mediated by phosphorothioate antisense oligonucleotides complementary to the gag region of the HIV-1 genome in vitro. Cell lysate experiments in H9 and U937 cells chronically infected with HIV-1 IIIB showed RNase H cleavage of unspliced gag message but no cleavage of spliced message which did not contain the target gag region. RNase H cleavage products were detected at oligonucleotide concentrations as low as 0.01 microM and the RNase H activity was seen to be concentration dependent. Similar experiments with 1-, 3- and 5-mismatch oligonucleotides demonstrated sequence specificity at low concentrations, with cleavage of gag mRNA correlating with the predicted activities of the parent and mismatch oligonucleotides based on their hybridization melting temperatures. Experiments in living cells suggested that RNase H-specific antisense activity was largely determined by the amount of oligonucleotide taken up by the different cell lines studied. RNase H cleavage products were detected in antisense oligonucleotide treated MT-4 cells acutely infected with HIV-1 IIIB, but not in infected H9 cells treated with oligonucleotide under the same conditions. The data presented demonstrate potent and specific RNase H cleavage of HIV-1 mRNA mediated by an antisense oligonucleotide targeted to HIV-1 gag mRNA, and are in agreement with previous reports that the major obstacle to demonstrating antisense activity in living cells remains the lack of penetration of these agents into the desired cellular compartment.