- A.A.M. Bokor (1)
- C. Geerts-Dimitriadou (1)
- R.W. Goldbach (1)
- J.A.L. Kan van (1)
- R.J.M. Kormelink (1)
- J. Oost van der (2)
- D.J. Patel (1)
- R.T.M. Poulter (1)
- Y. Rao (1)
- G. Sheng (1)
- D.C. Swarts (1)
- W. Tian (1)
- Y. Wang (1)
- J. Wang (1)
- H. Zhao (1)
- M.P. Zwart (1)
Structure-based cleavage mechanism of Thermus thermophilus Argonaute DNA guide strand-mediated DNA target cleavage
Sheng, G. ; Zhao, H. ; Wang, J. ; Rao, Y. ; Tian, W. ; Swarts, D.C. ; Oost, J. van der; Patel, D.J. ; Wang, Y. - \ 2014
Proceedings of the National Academy of Sciences of the United States of America 111 (2014)2. - ISSN 0027-8424 - p. 652 - 657.
crystal-structure - rna recognition - silencing complex - substrate-specificity - slicer activity - piwi protein - paz domain - human risc - endonuclease - interference
We report on crystal structures of ternary Thermus thermophilus Argonaute (TtAgo) complexes with 5'-phosphorylated guide DNA and a series of DNA targets. These ternary complex structures of cleavage-incompatible, cleavage-compatible, and postcleavage states solved at improved resolution up to 2.2 Å have provided molecular insights into the orchestrated positioning of catalytic residues, a pair of Mg2+ cations, and the putative water nucleophile positioned for in-line attack on the cleavable phosphate for TtAgo-mediated target cleavage by a RNase H-type mechanism. In addition, these ternary complex structures have provided insights into protein and DNA conformational changes that facilitate transition between cleavage-incompatible and cleavage-compatible states, including the role of a Glu finger in generating a cleavage-competent catalytic Asp-Glu-Asp-Asp tetrad. Following cleavage, the seed segment forms a stable duplex with the complementary segment of the target strand
Molecular biology - New tool for genome surgery
Oost, J. van der - \ 2013
Science 339 (2013)6121. - ISSN 0036-8075 - p. 768 - 770.
adaptive immunity - dna cleavage - rna - endonuclease - bacteria
Gene therapy is the holy grail of human medicine. Many diseases are caused by a defective gene, sometimes with a mutation as subtle as a single-nucleotide variation. Before restoration of such a mutation in a patient's genome can take place, the target nucleotide sequence has to be cleaved at a single position, out of 3 billion possibilities. This degree of precise surgery requires an enzyme with highly selective target recognition. Successful editing of eukaryotic genomes has been accomplished with DNA nucleases designed to bear a unique site that binds to a specific DNA sequence. A major drawback of these protein-guided systems to "engineer" genomes, however, is that each new target sequence requires laboriously adjusting the specificity of the nuclease's DNA binding site. On pages 819 and 823 of this issue, Cong et al. (1) and Mali et al. (2) describe efficient genome editing in human cells based on an RNA-guided system
Base-pairing promotes leader selection to prime in vitro influenza genome transcription
Geerts-Dimitriadou, C. ; Zwart, M.P. ; Goldbach, R.W. ; Kormelink, R.J.M. - \ 2011
Virology 409 (2011)1. - ISSN 0042-6822 - p. 17 - 26.
messenger-rna synthesis - cap-snatching mechanism - mosaic-virus rnas - viral-rna - 5' ends - complementary rna - a virus - polymerase - endonuclease - sequences
The requirements for alignment of capped leader sequences along the viral genome during influenza transcription initiation (cap-snatching) have long been an enigma. In this study, competition experiments using an in vitro transcription assay revealed that influenza virus transcriptase prefers leader sequences with base complementarity to the 3'-ultimate residues of the viral template, 10 or 11 nt from the 5' cap. Internal priming at the 3'-penultimate residue, as well as prime-and-realign was observed. The nucleotide identity immediately 5' of the base-pairing residues also affected cap donor usage. Application to the in vitro system of RNA molecules with increased base complementarity to the viral RNA template showed stronger reduction of globin RNA leader initiated influenza transcription compared to those with a single base-pairing possibility. Altogether the results indicated an optimal cap donor consensus sequence of 7mG-(N)7–8-(A/U/G)-(A/U)-AGC-3'.
Sexual mating of Botrytis cinerea illustrates PRP8 intein HEG activity
Bokor, A.A.M. ; Kan, J.A.L. van; Poulter, R.T.M. - \ 2010
Fungal Genetics and Biology 47 (2010)4. - ISSN 1087-1845 - p. 392 - 398.
saccharomyces-cerevisiae - branched intermediate - gene conversion - selfish gene - endonuclease - populations
Strains of Botrytis cinerea are polymorphic for the presence of an intein in the Prp8 gene (intein +/-). The intein encodes a homing endonuclease (HEG). During meiosis in an intein +/- heterozygote, the homing endonuclease initiates intein ‘homing’ by inducing gene conversion. In such meioses, the homing endonuclease triggers gene conversion of the intein together with its flanking sequences into the empty allele. The efficiency of gene conversion of the intein was found to be 100%. The extent of flanking sequence affected by the gene conversion varied in different meioses. A survey of the inteins and flanking sequences of a group B. cinerea isolates indicates that there are two distinct variants of the intein both of which have active HEGs. The survey also suggests that the intein has been actively homing during the evolution of the species and that the PRP8 intein may have entered the species by horizontal transfer