Dataset Information

DNA recognition and the precleavage state during single-stranded DNA transposition in D. radiodurans.

ABSTRACT: Bacterial insertion sequences (ISs) from the IS200/IS605 family encode the smallest known DNA transposases and mobilize through single-stranded DNA transposition. Transposition by one particular family member, ISDra2 from Deinococcus radiodurans, is dramatically stimulated upon massive ? irradiation. We have determined the crystal structures of four ISDra2 transposase/IS end complexes; combined with in vivo activity assays and fluorescence anisotropy binding measurements, these have revealed the molecular basis of strand discrimination and transposase action. The structures also show that previously established structural rules of target site recognition that allow different specific sequences to be targeted are only partially conserved among family members. Furthermore, we have captured a fully assembled active site including the scissile phosphate bound by a divalent metal ion cofactor (Cd²(+)) that supports DNA cleavage. Finally, the observed active site rearrangements when the transposase binds a metal ion in which it is inactive provide a clear rationale for metal ion specificity.

SUBMITTER: Hickman AB

PROVIDER: S-EPMC2989099 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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DNA recognition and the precleavage state during single-stranded DNA transposition in D. radiodurans.

Hickman Alison Burgess AB James Jeffrey A JA Barabas Orsolya O Pasternak Cécile C Ton-Hoang Bao B Chandler Michael M Sommer Suzanne S Dyda Fred F

The EMBO journal 20101001 22

Bacterial insertion sequences (ISs) from the IS200/IS605 family encode the smallest known DNA transposases and mobilize through single-stranded DNA transposition. Transposition by one particular family member, ISDra2 from Deinococcus radiodurans, is dramatically stimulated upon massive γ irradiation. We have determined the crystal structures of four ISDra2 transposase/IS end complexes; combined with in vivo activity assays and fluorescence anisotropy binding measurements, these have revealed the ...[more]

PMID: 20890269

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Project description:Deinococcus radiodurans R1 is one of the most radiation-resistant organisms known and is able to repair an unusually large amount of DNA damage without induced mutation. Single-stranded DNA-binding (SSB) protein is an essential protein in all organisms and is involved in DNA replication, recombination and repair. The published genomic sequence from Deinococcus radiodurans includes a putative single-stranded DNA-binding protein gene (ssb; DR0100) requiring a translational frameshift for synthesis of a complete SSB protein. The apparently tripartite gene has inspired considerable speculation in the literature about potentially novel frameshifting or RNA editing mechanisms. Immediately upstream of the ssb gene is another gene (DR0099) given an ssb-like annotation, but left unexplored.A segment of the Deinococcus radiodurans strain R1 genome encompassing the ssb gene has been re-sequenced, and two errors involving omitted guanine nucleotides have been documented. The corrected sequence incorporates both of the open reading frames designated DR0099 and DR0100 into one contiguous ssb open reading frame (ORF). The corrected gene requires no translational frameshifts and contains two predicted oligonucleotide/oligosaccharide-binding (OB) folds. The protein has been purified and its sequence is closely related to the Thermus thermophilus and Thermus aquaticus SSB proteins. Like the Thermus SSB proteins, the SSBDr functions as a homodimer. The Deinococcus radiodurans SSB homodimer stimulates Deinococcus radiodurans RecA protein and Escherichia coli RecA protein-promoted DNA three-strand exchange reactions with at least the same efficiency as the Escherichia coli SSB homotetramer.The correct Deinococcus radiodurans ssb gene is a contiguous open reading frame that codes for the largest bacterial SSB monomer identified to date. The Deinococcus radiodurans SSB protein includes two OB folds per monomer and functions as a homodimer. The Deinococcus radiodurans SSB protein efficiently stimulates Deinococcus radiodurans RecA and also Escherichia coli RecA protein-promoted DNA strand exchange reactions. The identification and purification of Deinococcus radiodurans SSB protein not only allows for greater understanding of the SSB protein family but provides an essential yet previously missing player in the current efforts to understand the extraordinary DNA repair capacity of Deinococcus radiodurans.

Project description:UnlabelledNeisseria gonorrhoeae is naturally competent for transformation. The first step of the transformation process is the uptake of DNA from the environment into the cell. This transport step is driven by a powerful molecular machine. Here, we addressed the question whether this machine imports single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) at similar rates. The fluorescence signal associated with the uptake of short DNA fragments labeled with a single fluorescent marker molecule was quantified. We found that ssDNA with a double-stranded DNA uptake sequence (DUS) was taken up with a similar efficiency as dsDNA. Imported ssDNA was degraded rapidly, and the thermonuclease Nuc was required for degradation. In a nuc deletion background, dsDNA and ssDNA with a double-stranded DUS were imported and used as the substrates for transformation, whereas the import and transformation efficiencies of ssDNA with single-stranded DUS were below the detection limits. We conclude that the DNA uptake machine requires a double-stranded DUS for efficient DNA recognition and transports ssDNA and dsDNA with comparable efficiencies.ImportanceBacterial transformation enables bacteria to exchange genetic information. It can speed up adaptive evolution and enhances the potential of DNA repair. The transport of DNA through the outer membrane is the first step of transformation in Gram-negative species. It is driven by a powerful molecular machine whose mechanism remains elusive. Here, we show for Neisseria gonorrhoeae that the machine transports single- and double-stranded DNA at comparable rates, provided that the species-specific DNA uptake sequence is double stranded. Moreover, we found that single-stranded DNA taken up into the periplasm is rapidly degraded by the thermonuclease Nuc. We conclude that the secondary structure of transforming DNA is important for the recognition of self DNA but not for the process of transport through the outer membrane.

Dataset Information

DNA recognition and the precleavage state during single-stranded DNA transposition in D. radiodurans.

Publications

DNA recognition and the precleavage state during single-stranded DNA transposition in D. radiodurans.

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