Structure of yeast Argonaute with guide RNA

The RNA-induced silencing complex, comprising Argonaute and guide RNA, mediates RNA interference. Here we report the 3.2 Å crystal structure of Kluyveromyces polysporus Argonaute (KpAGO) fortuitously complexed with guide RNA originating from small-RNA duplexes autonomously loaded and processed by recombinant KpAGO. Despite their diverse sequences, guide-RNA nucleotides 1–8 are positioned similarly, with sequence-independent contacts to bases, phosphates and 2′-hydroxyl groups pre-organizing the backbone of nucleotides 2–8 in a near-A-form conformation. Compared with prokaryotic Argonautes, KpAGO has numerous surface-exposed insertion segments, with a cluster of conserved insertions repositioning the N domain to enable full propagation of guide–target pairing. Compared with Argonautes in inactive conformations, KpAGO has a hydrogen-bond network that stabilizes an expanded and repositioned loop, which inserts an invariant glutamate into the catalytic pocket. Mutation analyses and analogies to ribonuclease H indicate that insertion of this glutamate finger completes a universally conserved catalytic tetrad, thereby activating Argonaute for RNA cleavage.National Institutes of Health (U.S.) (grant AI068776)National Institutes of Health (U.S.) (grant GM61835)Human Frontier Science Program (Strasbourg, France) (Long-term Fellowship)Japan Society for the Promotion of ScienceHoward Hughes Medical Institute (Investigator)National Science Foundation (U.S.) (Graduate Research Fellowship

Base Sequence Context Effects on Nucleotide Excision Repair

Nucleotide excision repair (NER) plays a critical role in maintaining the integrity of the genome when damaged by bulky DNA lesions, since inefficient repair can cause mutations and human diseases notably cancer. The structural properties of DNA lesions that determine their relative susceptibilities to NER are therefore of great interest. As a model system, we have investigated the major mutagenic lesion derived from the environmental carcinogen benzo[a]pyrene (B[a]P), 10S (+)-trans-anti-B[a]P-N2-dG in six different sequence contexts that differ in how the lesion is positioned in relation to nearby guanine amino groups. We have obtained molecular structural data by NMR and MD simulations, bending properties from gel electrophoresis studies, and NER data obtained from human HeLa cell extracts for our six investigated sequence contexts. This model system suggests that disturbed Watson-Crick base pairing is a better recognition signal than a flexible bend, and that these can act in concert to provide an enhanced signal. Steric hinderance between the minor groove-aligned lesion and nearby guanine amino groups determines the exact nature of the disturbances. Both nearest neighbor and more distant neighbor sequence contexts have an impact. Regardless of the exact distortions, we hypothesize that they provide a local thermodynamic destabilization signal for repair

Non-CG methylation patterns shape the epigenetic landscape in Arabidopsis.

DNA methylation occurs in CG and non-CG sequence contexts. Non-CG methylation is abundant in plants and is mediated by CHROMOMETHYLASE (CMT) and DOMAINS REARRANGED METHYLTRANSFERASE (DRM) proteins; however, its roles remain poorly understood. Here we characterize the roles of non-CG methylation in Arabidopsis thaliana. We show that a poorly characterized methyltransferase, CMT2, is a functional methyltransferase in vitro and in vivo. CMT2 preferentially binds histone H3 Lys9 (H3K9) dimethylation and methylates non-CG cytosines that are regulated by H3K9 methylation. We revealed the contributions and redundancies between each non-CG methyltransferase in DNA methylation patterning and in regulating transcription. We also demonstrate extensive dependencies of small-RNA accumulation and H3K9 methylation patterning on non-CG methylation, suggesting self-reinforcing mechanisms between these epigenetic factors. The results suggest that non-CG methylation patterns are critical in shaping the landscapes of histone modification and small noncoding RNA

Mutagenic nucleotide incorporation and hindered translocation by a food carcinogen C8-dG adduct in Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4): modeling and dynamics studies

Bulky carcinogen-DNA adducts commonly cause replicative polymerases to stall, leading to a switch to bypass polymerases. We have investigated nucleotide incorporation opposite the major adduct of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in the DinB family polymerase, Dpo4, using molecular modeling and molecular dynamics (MD) simulations. PhIP, the most prevalent heterocyclic aromatic amine formed by cooking of proteinaceous food, is mutagenic in mammalian cells and is implicated in mammary and colon tumors. Our results show that the dG-C8-PhIP adduct can be accommodated in the spacious major groove Dpo4 open pocket, with Dpo4 capable of incorporating dCTP, dTTP or dATP opposite the adduct reasonably well. However, the PhIP ring system on the minor groove side would seriously disturb the active site, regardless of the presence and identity of dNTP. Furthermore, the simulations indicate that dATP and dTTP are better incorporated in the damaged system than in their respective mismatched but unmodified controls, suggesting that the PhIP adduct enhances incorporation of these mismatches. Finally, bulky C8-dG adducts, situated in the major groove, are likely to impede translocation in this polymerase (Rechkoblit et al. (2006), PLoS Biol., 4, e11). However, N(2)-dG adducts, which can reside on the minor groove side, appear to cause less hindrance when in this position

Comparative NMR study of A_n-bulge loops in DNA duplexes: intrahelical stacking of A, A-A, and A-A-A bulge loops

We have prepared a series of deoxyoligonucleotide duplexes of the sequence d(G-C-A-T-C-G-X-G-C-T-A-C-G)•d(C-G-T-A-G-C-C-G-T-C), in which X represents either one (A), two (A-A), or three (A-A-A) unpaired adenine bases. Using two-dimensional proton and phosphorus NMR spectroscopy, we have characterized conformational features of these bulge-loop duplexes in solution. We find that Watson-Crick hydrogen bonding is intact for all 12 base pairs, including the GC bases that flank the bulge loop. Observation of NOE connectivities in both H_2O and D_2O allows us to unambiguously localize all of the bulged adenine residues to intrahelical positions within the duplex. This is in contrast to an earlier model for multiple-base bulge loops in DNA [Bhattacharyya, A., & Lilley, D. M. J. (1989) Nucleic Acids Res. 17, 6821-68401, in which all but the most 5’ bulged base are looped out into solution. We find that insertion of two or three bases into the duplex results in the disruption of specific sequential NOEs for the base step across from the bulge loop site on the opposite strand. This disruption is characterized by a partial shearing apart of these bases, such that certain sequential NOEs for this base step are preserved. We observe a downfield-shifted phosphorus resonance, which we assign in the A-A-A bulge duplex to the 3‘ side of the last bulged adenine residue. Proton and phosphorus chemical shift trends within the A,-bulge duplex series indicate that there is an additive effect on the structural perturbations caused by additional unpaired bases within the bulge loop. This finding parallels previous observations [Bhattacharyya, A., & Lilley, D. M. J. (1989) Nucleic Acids Res. 17, 6821-6840; Hsieh, C.-H., & Griffith, J. D. (1989) Proc. Nutl. Acud. Sci. U.S.A. 86,4833-48371 on the magnitude of the induced bending of DNA duplexes by multiple-base bulge loops

Comparative NMR study of A_n-bulge loops in DNA duplexes: intrahelical stacking of A, A-A, and A-A-A bulge loops

We have prepared a series of deoxyoligonucleotide duplexes of the sequence d(G-C-A-T-C-G-X-G-C-T-A-C-G)•d(C-G-T-A-G-C-C-G-T-C), in which X represents either one (A), two (A-A), or three (A-A-A) unpaired adenine bases. Using two-dimensional proton and phosphorus NMR spectroscopy, we have characterized conformational features of these bulge-loop duplexes in solution. We find that Watson-Crick hydrogen bonding is intact for all 12 base pairs, including the GC bases that flank the bulge loop. Observation of NOE connectivities in both H_2O and D_2O allows us to unambiguously localize all of the bulged adenine residues to intrahelical positions within the duplex. This is in contrast to an earlier model for multiple-base bulge loops in DNA [Bhattacharyya, A., & Lilley, D. M. J. (1989) Nucleic Acids Res. 17, 6821-68401, in which all but the most 5’ bulged base are looped out into solution. We find that insertion of two or three bases into the duplex results in the disruption of specific sequential NOEs for the base step across from the bulge loop site on the opposite strand. This disruption is characterized by a partial shearing apart of these bases, such that certain sequential NOEs for this base step are preserved. We observe a downfield-shifted phosphorus resonance, which we assign in the A-A-A bulge duplex to the 3‘ side of the last bulged adenine residue. Proton and phosphorus chemical shift trends within the A,-bulge duplex series indicate that there is an additive effect on the structural perturbations caused by additional unpaired bases within the bulge loop. This finding parallels previous observations [Bhattacharyya, A., & Lilley, D. M. J. (1989) Nucleic Acids Res. 17, 6821-6840; Hsieh, C.-H., & Griffith, J. D. (1989) Proc. Nutl. Acud. Sci. U.S.A. 86,4833-48371 on the magnitude of the induced bending of DNA duplexes by multiple-base bulge loops

Accommodation of Helical Imperfections in Rhodobacter sphaeroides Argonaute Ternary Complexes with Guide RNA and Target DNA

Prokaryotic Argonaute (Ago) proteins were recently shown to target foreign genetic elements, thus making them a perfect model for studies of interference mechanisms. Here, we study interactions of Rhodobacter sphaeroides Ago (RsAgo) with guide RNA (gRNA) and fully complementary or imperfect target DNA (tDNA) using biochemical and structural approaches. We show that RsAgo can specifically recognize both the first nucleotide in gRNA and complementary nucleotide in tDNA, and both interactions contribute to nucleic acid binding. Non-canonical pairs and bulges on the target strand can be accommodated by RsAgo with minimal perturbation of the duplex but significantly reduce RsAgo affinity to tDNA. Surprisingly, mismatches between gRNA and tDNA induce dissociation of the guide-target duplex from RsAgo. Our results reveal plasticity in the ability of Ago proteins to accommodate helical imperfections, show how this might affect the efficiency of RNA silencing, and suggest a potential mechanism for guide release and Ago recycling

Modeling cancer genomic data in yeast reveals selection against ATM function during tumorigenesis

The DNA damage response (DDR) comprises multiple functions that collectively preserve genomic integrity and suppress tumorigenesis. The Mre11 complex and ATM govern a major axis of the DDR and several lines of evidence implicate that axis in tumor suppression. Components of the Mre11 complex are mutated in approximately five percent of human cancers. Inherited mutations of complex members cause severe chromosome instability syndromes, such as Nijmegen Breakage Syndrome, which is associated with strong predisposition to malignancy. And in mice, Mre11 complex mutations are markedly more susceptible to oncogene- induced carcinogenesis. The complex is integral to all modes of DNA double strand break (DSB) repair and is required for the activation of ATM to effect DNA damage signaling. To understand which functions of the Mre11 complex are important for tumor suppression, we undertook mining of cancer genomic data from the clinical sequencing program at Memorial Sloan Kettering Cancer Center, which includes the Mre11 complex among the 468 genes assessed. Twenty five mutations in MRE11 and RAD50 were modeled in S. cerevisiae and in vitro. The mutations were chosen based on recurrence and conservation between human and yeast. We found that a significant fraction of tumor-borne RAD50 and MRE11 mutations exhibited separation of function phenotypes wherein Tel1/ATM activation was severely impaired while DNA repair functions were mildly or not affected. At the molecular level, the gene products of RAD50 mutations exhibited defects in ATP binding and hydrolysis. The data reflect the importance of Rad50 ATPase activity for Tel1/ATM activation and suggest that inactivation of ATM signaling confers an advantage to burgeoning tumor cells