The mutY homolog gene (mutY(Dr)) from Deinococcus radiodurans encodes a 39.4-kDa protein consisting of 363 amino acids that displays 35% identity to the Escherichia coli MutY (MutY(Ec)) protein. Expressed MutY(Dr) is able to complement E. coli mutY mutants but not mutM mutants to reduce the mutation frequency. The glycosylase and binding activities of MutY(Dr) with an A/G-containing substrate are more sensitive to high salt and EDTA concentrations than the activities with an A/7,8-dihydro-8-oxoguanine (GO)-containing substrate are. Like the MutY(Ec) protein, purified recombinant MutY(Dr) expressed in E. coli has adenine glycosylase activity with A/G, A/C, and A/GO mismatches and weak guanine glycosylase activity with a G/GO mismatch. However, MutY(Dr) exhibits limited apurinic/apyrimidinic lyase activity and can form only weak covalent protein-DNA complexes in the presence of sodium borohydride. This may be due to an arginine residue that is present in MutY(Dr) at the position corresponding to the position of MutY(Ec) Lys142, which forms the Schiff base with DNA. The kinetic parameters of MutY(Dr) are similar to those of MutY(Ec). Although MutY(Dr) has similar substrate specificity and a binding preference for an A/GO mismatch over an A/G mismatch, as MutY(Ec) does, the binding affinities for both mismatches are slightly lower for MutY(Dr) than for MutY(Ec). Thus, MutY(Dr) can protect the cell from GO mutational effects caused by ionizing radiation and oxidative stress.

Deinococcus radiodurans strain rec30, which is a DNA damage repair-deficient mutant, has been estimated to be defective in the deinococcal recA gene. To identify the mutation site of strain rec30 and obtain information about the region flanking the gene, a 4.4-kb fragment carrying the wild-type recA gene was sequenced. It was revealed that the recA locus forms a polycistronic operon with the preceding cistrons (orf105a and orf105b). Predicted amino acid sequences of orf105a and orf105b showed substantial similarity to the competence-damage inducible protein (cinA gene product) from Streptococcus pneumoniae and the 2'-5' RNA ligase from Escherichia coli, respectively. By analyzing polymerase chain reaction (PCR) fragments derived from the genomic DNA of strain rec30, the mutation site in the strain was identified as a single G:C to A:T transition which causes an amino acid substitution at position 224 (Gly to Ser) of the deinococcal RecA protein. Furthermore, we succeeded in expressing both the wild-type and mutant recA genes of D. radiodurans in E. coli without any obvious toxicity or death. The gamma-ray resistance of an E. coli recA1 strain was fully restored by the expression of the wild-type recA gene of D. radiodurans that was cloned in an E. coli vector plasmid. This result is consistent with evidence that RecA proteins from many bacterial species can functionally complement E. coli recA mutants. In contrast with the wild-type gene, the mutant recA gene derived from strain rec30 did not complement E. coli recA1, suggesting that the mutant RecA protein lacks functional activity for recombinational repair.

We isolated a radiosensitive mutant strain, KR4128, from a wild-type strain of Deinococcus radiodurans, which is known as a extremely radioresistant bacterium. The gene that restore the defect of the mutant in DNA repair was cloned, and it turned out to be the homolog of the recN gene of Escherichia coli. The recN gene encoded a protein of 58 kDa, and, in its N-terminal region, a potential ATP binding domain was conserved as expected for a prokaryotic RecN protein. An analysis of sequence of the mutant recN gene revealed a G:C to T:A transversion near the 3' end of the coding region. This alteration causes an ochre mutation, and results in the truncation of 47 amino acids from the C-terminal region of the RecN protein. The null mutant of recN gene was constructed by insertional mutagenesis, and it showed substantial sensitivities to various types of DNA damaging agents, indicating that a single defect in the recN gene can directly affect the DNA damage resistant phenotype in D. radiodurans. The recN locus of KR4128 was also disrupted and the disruptant indicated the sensitivity that was indistinguishable from its progenitor. The result indicate that the transversion in the recN gene of KR4128 cells causes a complete loss of function of the RecN protein and thus the C-terminal region of the RecN protein includes domain essential to its function.

A gene encoding homoisocitrate dehydrogenase (HICDH) of Deinococcus radiodurans was cloned, sequenced, and overexpressed in Escherichia coli. The amino acid sequence was 63% identical to HICDH from Thermus thermophilus (Tth-HICDH). Similar to Tth-HICDH, purified, recombinant Dra-HICDH was a tetramer, required K+ and Mn2+ for activity, and used NAD+ as a coenzyme. However, unlike Tth-HICDH, which has a 20-fold preference for isocitrate over homoisocitrate, Dra-HICDH preferred homoisocitrate to isocitrate by 1.5-fold. Moreover, it catalyzed the oxidation of 3-isopropylmalate, albeit at approximately 0.1% the rate seen with homoisocitrate and isocitrate. Saturation mutagenesis of Dra-HICDH Arg87 was next performed because an orthologous Arg85 to valine mutation in Tth-HICDH results in loss of activity toward isocitrate, but in retention of activity toward homoisocitrate. Unexpectedly, the Arg85Val variant became able to catalyze the oxidation of 3-isopropylmalate. Screening of the saturation mutagenesis library identified two variants, Arg87Val and Arg87Thr, that were able to catalyze the oxidation of homoisocitrate, but not isocitrate or 3-isopropylmalate. Deletion of Dra-HICDH Ala80, a residue missing from Tth-HICDH and predicted to reside at the entrance of alpha-helix Arg87, resulted in alterations in substrate specificity that rendered Dra-HICDH similar to Tth-HICDH; i.e., a 4-fold preference for isocitrate over homoisocitrate and inability to catalyze the oxidation of 3-isopropylmalate. Seemingly minor changes in primary sequence result in changes in substrate specificity of beta-decarboxylating dehydrogenases.

tmRNA combines tRNA- and mRNA-like properties and ameliorates problems arising from stalled ribosomes. Research on the mechanism, structure and biology of tmRNA is served by the tmRNA website (http://www.indiana.edu/~ tmrna), a collection of sequences, alignments, secondary structures and other information. Because many of these sequences are not in GenBank, a BLAST server has been added; another new feature is an abbreviated alignment for the tRNA-like domain only. Many tmRNA sequences from plastids have been added, five found in public sequence data and another 10 generated by direct sequencing; detection in early-branching members of the green plastid lineage brings coverage to all three primary plastid lineages. The new sequences include the shortest known tmRNA sequence. While bacterial tmRNAs usually have a lone pseudoknot upstream of the mRNA segment and a string of three or four pseudoknots downstream, plastid tmRNAs collectively show loss of pseudoknots at both postions. The pseudoknot-string region is also too short to contain the usual pseudoknot number in another new entry, the tmRNA sequence from a bacterial endosymbiont of insect cells, Tremblaya princeps. Pseudoknots may optimize tmRNA function in free-living bacteria, yet become dispensible when the endosymbiotic lifestyle relaxes selective pressure for fast growth.

The SMC proteins are found in nearly all living organisms examined, where they play crucial roles in mitotic chromosome dynamics, regulation of gene expression, and DNA repair. We have explored the phylogenetic relationships of SMC proteins from prokaryotes and eukaryotes, as well as their relationship to similar ABC ATPases, using maximum-likelihood analyses. We have also investigated the coevolution of different domains of eukaryotic SMC proteins and attempted to account for the evolutionary patterns we have observed in terms of available structural data. Based on our analyses, we propose that each of the six eukaryotic SMC subfamilies originated through a series of ancient gene duplication events, with the condensins evolving more rapidly than the cohesins. In addition, we show that the SMC5 and SMC6 subfamily members have evolved comparatively rapidly and suggest that these proteins may perform redundant functions in higher eukaryotes. Finally, we propose a possible structure for the SMC5/SMC6 heterodimer based on patterns of coevolution.

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 (Cd?(+)) 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.

In Deinococcus radiodurans, the extreme resistance to DNA-shattering treatments such as ionizing radiation or desiccation is correlated with its ability to reconstruct a functional genome from hundreds of chromosomal fragments. The rapid reconstitution of an intact genome is thought to occur through an extended synthesis-dependent strand annealing process (ESDSA) followed by DNA recombination. Here, we investigated the role of key components of the RecF pathway in ESDSA in this organism naturally devoid of RecB and RecC proteins. We demonstrate that inactivation of RecJ exonuclease results in cell lethality, indicating that this protein plays a key role in genome maintenance. Cells devoid of RecF, RecO, or RecR proteins also display greatly impaired growth and an important lethal sectoring as bacteria devoid of RecA protein. Other aspects of the phenotype of recFOR knock-out mutants paralleled that of a DeltarecA mutant: DeltarecFOR mutants are extremely radiosensitive and show a slow assembly of radiation-induced chromosomal fragments, not accompanied by DNA synthesis, and reduced DNA degradation. Cells devoid of RecQ, the major helicase implicated in repair through the RecF pathway in E. coli, are resistant to gamma-irradiation and have a wild-type DNA repair capacity as also shown for cells devoid of the RecD helicase; in contrast, DeltauvrD mutants show a markedly decreased radioresistance, an increased latent period in the kinetics of DNA double-strand-break repair, and a slow rate of fragment assembly correlated with a slow rate of DNA synthesis. Combining RecQ or RecD deficiency with UvrD deficiency did not significantly accentuate the phenotype of DeltauvrD mutants. In conclusion, RecFOR proteins are essential for DNA double-strand-break repair through ESDSA whereas RecJ protein is essential for cell viability and UvrD helicase might be involved in the processing of double stranded DNA ends and/or in the DNA synthesis step of ESDSA.

The type II restriction endonucleases form one of the largest families of biochemically-characterized proteins. These endonucleases typically share little sequence similarity, except among isoschizomers that recognize the same sequence. MmeI is an unusual type II restriction endonuclease that combines endonuclease and methyltransferase activities in a single polypeptide. MmeI cuts DNA 20 bases from its recognition sequence and modifies just one DNA strand for host protection. Using MmeI as query we have identified numerous putative genes highly similar to MmeI in database sequences. We have cloned and characterized 20 of these MmeI homologs. Each cuts DNA at the same distance as MmeI and each modifies a conserved adenine on only one DNA strand for host protection. However each enzyme recognizes a unique DNA sequence, suggesting these enzymes are undergoing rapid evolution of DNA specificity. The MmeI family thus provides a rich source of novel endonucleases while affording an opportunity to observe the evolution of DNA specificity. Because the MmeI family enzymes employ modification of only one DNA strand for host protection, unlike previously described type II systems, we propose that such single-strand modification systems be classified as a new subgroup, the type IIL enzymes, for Lone strand DNA modification.

The resection of DNA strand with a 5? end at double-strand breaks is an essential step in recombinational DNA repair. RecJ, a member of DHH family proteins, is the only 5? nuclease involved in the RecF recombination pathway. Here, we report the crystal structures of Deinococcus radiodurans RecJ in complex with deoxythymidine monophosphate (dTMP), ssDNA, the C-terminal region of single-stranded DNA-binding protein (SSB-Ct) and a mechanistic insight into the RecF pathway. A terminal 5?-phosphate-binding pocket above the active site determines the 5?-3? polarity of the deoxy-exonuclease of RecJ; a helical gateway at the entrance to the active site admits ssDNA only; and the continuous stacking interactions between protein and nine nucleotides ensure the processive end resection. The active site of RecJ in the N-terminal domain contains two divalent cations that coordinate the nucleophilic water. The ssDNA makes a 180�X turn at the scissile phosphate. The C-terminal domain of RecJ binds the SSB-Ct, which explains how RecJ and SSB work together to efficiently process broken DNA ends for homologous recombination.

RNase J is a conserved ribonuclease that belongs to the �]-CASP family of nucleases. It possesses both endo- and exo-ribonuclease activities, which play a key role in pre-rRNA maturation and mRNA decay. Here we report high-resolution crystal structures of Deinococcus radiodurans RNase J complexed with RNA or uridine 5'-monophosphate in the presence of manganese ions. Biochemical and structural studies revealed that RNase J uses zinc ions for two-metal-ion catalysis. One residue conserved among RNase J orthologues (motif B) forms specific electrostatic interactions with the scissile phosphate of the RNA that is critical for the catalysis and product stabilization. The additional manganese ion, which is coordinated by conserved residues at the dimer interface, is critical for RNase J dimerization and exonuclease activity. The structures may also shed light on the mechanism of RNase J exo- and endonucleolytic activity switch.

Deinococcus radiodurans survives extreme doses of radiations contributed by efficient DNA repair pathways. DR2417 (DncA) was detected separately both in a pool of nucleotide binding proteins and multiprotein complex isolated from cells undergoing DNA repair. DR_2417m ORF was sequenced and amino acid sequence of DncA was search for structural similarities with other proteins and functional motifs. Recombinant DncA was characterized for its DNA metabolic functions in vitro and its role in radiation resistance. Sequencing of DR_2417m did not show the reported frame shift at 996th nucleotide position of this gene. DncA showed similarities with �]-CASP family nucleases. Recombinant protein acted efficiently on dsDNA and showed an Mn2+ dependent 3'��5' exonuclease and ssDNA/dsDNA junction endonuclease activities while a very low level activity on RNA. The DNase activity of this protein was inhibited in presence of ATP. Its transcription was induced upon �^ radiation exposure and a reduction in its copy number resulted in reduced growth rate and loss of �^ radiation resistance in Deinococcus. Our results suggest that DncA was a novel nuclease of �] CASP family having a strong dsDNA end processing activity and it seems to be an essential gene required for both growth and �^ radiation resistance of this bacterium. Traditionally DncA should have shown both DNase and RNase functions as other members of �] CASP family nucleases. A strong DNase and poor RNase activity possibly made it functionally significant in the radioresistance of D. radiodurans, which would be worth investigating independently.

Deinococcus radiodurans (Dr) possesses a prominent ability to repair the DNA injury induced by various DNA-damaging agents including mitomycin C (MC), ultraviolet light (UV) and ionizing radiation. DNA damage resistance was restored in MC sensitive (MC(S)) mutants 2621 and 3021 by transforming with DNAs of four cosmid clones derived from the gene library of strain KD8301, which showed wild type (wt) phenotype to DNA-damaging agents. Gene affected by mutation (mtcA or mtcB) in both mutants was cloned and its nucleotide (nt) sequence was determined. The deduced amino acid (aa) sequence of the gene product consists of 1016 aa and shares homology with many bacterial UvrA proteins. The mutation sites of both mutants were identified by analyzing the polymerase chain reaction (PCR) fragments derived from the genomic DNA of the mutants. A 144-base pair (bp) deletion including the start codon for the uvrA gene was observed in DNA of the mutant 3021, causing a defect in the gene. On the other hand, an insertion sequence (IS) element intervened in the uvrA gene of the mutant 2621, suggesting the insertional inactivation of the gene. The IS element comprises 1322-bp long, flanked by 19-bp inverted terminal repeats (ITR), and generated a 6-bp target duplication (TD). Two open reading frames (ORFs) were found in the IS element. The deduced aa sequences of large and small ORFs show homology to a putative transposase found in IS4 of Escherichia coli (Ec) and to a resolvase found in ISXc5 of Xanthomonas campestris (Xc), respectively. This is the first discovery of IS element in deinobacteria, and the IS element was designated IS2621.

The complete nucleotide sequence of the gene encoding the surface (hexagonally packed intermediate [HPI])-layer polypeptide of Deinococcus radiodurans Sark was determined and found to encode a polypeptide of 1,036 amino acids. Amino acid sequence analysis of about 30% of the residues revealed that the mature polypeptide consists of at least 978 amino acids. The N terminus was blocked to Edman degradation. The results of proteolytic modification of the HPI layer in situ and Mr estimations of the HPI polypeptide expressed in Escherichia coli indicated that there is a leader sequence. The N-terminal region contained a very high percentage (29%) of threonine and serine, including a cluster of nine consecutive serine or threonine residues, whereas a stretch near the C terminus was extremely rich in aromatic amino acids (29%). The protein contained at least two disulfide bridges, as well as tightly bound reducing sugars and fatty acids.

2',3'-Cyclic phosphodiesterase (CPDase) homologues have been found in all domains of life and are involved in diverse RNA and nucleotide metabolisms. The CPDase from Deinococcus radiodurans was crystallized and the crystals diffracted to 1.6 ? resolution, which is the highest resolution currently known for a CPDase structure. Structural comparisons revealed that the enzyme is in an open conformation in the absence of substrate. Nevertheless, the active site is well formed, and the representative motifs interact with sulfate ion, which suggests a conserved catalytic mechanism.