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Clock karser ks 203 инструкция

Apparent equilibrium dissociation constants KDs were calculated from binding curves plotted using fluorescence anisotropy against protein concentration. Sso bound ssDNA and dsDNA with apparent KDs of nM Clock karser ks 203 инструкция 2. By contrast, RadA bound ssDNA and dsDNA with apparent KDs of 2.

The ability of the aRadC proteins to bind tightly to ssDNA deserves further investigation. Since the L1 and L2 loops of the Clock karser ks 203 инструкция family seem to have a less basic character than those of RadA, the interaction may involve the intercalation of hydrophobic residues between DNA bases as is seen in SSB proteins as well as ionic interactions with the phosphodiester backbone.

Structural biology of SSo Structural comparison of Sso to other RecA superfamily proteins. Sso shares the highest structural similarity with the structure used for molecular replacement, the aRadC family member Pho from P.

However, Sso is not an active recombinase in vitro, and inhibits D-loop formation by RadA. We present the high-resolution crystal structure of Sso, which reveals key structural differences from the canonical RecA family recombinases that may explain its functional properties.

The structure has been submitted to the Protein Data Bank Clock karser ks 203 инструкция PDB code 2w0n. Crystallographic data and refinement. Despite the fact that there was no pyrophosphate or a compound with a similar atomic arrangement Clock karser ks 203 инструкция the crystallization or cryoprotection solution, there was a large area of elongated difference density with a clear tetragonal arrangement at one end, and a slightly less-well defined tetragonal arrangement at the other.

This density is in the proximity of a Walker motif, in the area occupied by the phosphate backbone of ADP in the 2dr3 structure. This density has been modelled as a pyrophosphate moiety, on the assumption that the molecule is endogenous E. RNA was prepared from S.

These data suggest that all three aradC genes are transcribed under normal growth conditions. Relative abundance of mRNA transcripts encoding RadA and paralogs in S. We and others have shown previously by global microarray analysis and Western blotting that radA expression is not induced appreciably by UV irradiation 25 ; However, Sheng and coworkers 23 reported a two-fold increase in S.

Given the significantly higher ssDNA binding affinity of Sso compared to RadA, it is unsurprising that the former may sequester ssDNA and thus prevent the formation of a RadA nucleoprotein filament as does SSB. However, the observation that Sso prevents a preformed ssDNA:RadA presynaptic filament from completing strand exchange suggests the possibility of an active disassembly process catalysed by Sso The regulation of the initiation of homologous recombination in eukaryotes is important for genome stability and the aRadC paralogs may perform a similar function.

This suggests Clock karser ks 203 инструкция Sso may displace RadA from RadA:ssDNA nucleoprotein filaments, either due simply to the higher ssDNA binding affinity of Sso or due to an active nucleofilament disassembly process. To mimic the in vivo environment, saturating concentrations of S. SSB inhibited D-loop formation by RadA Figure 4Dconsistent with studies in bacteria and eukarya 10 ; 32 ; To test whether Sso could play a role in displacing SSB to facilitate RadA access to DNA, we introduced pre-incubated DNA with SSB, followed by RadA, and finally Sso molar ratio of 1 SSB:5 RadA:5 Sso Figure 4D.

Neither reaction was completely ATP-dependent, an observation that is consistent with the behaviour of RecA family proteins when catalysing strand exchange reactions between relatively short DNA sequences As a further control, we tested the ability of the S. In contrast, the single-stranded DNA binding protein SSB from S. These data suggest that proteins such as Sso, RadA and Alba1 that can bind to both Clock karser ks 203 инструкция and dsDNA can promote limited strand exchange, potentially by passive equilibrium binding Clock karser ks 203 инструкция all the species present.

A phylogenetic analysis reveals that these are monophyletic, with robust bootstrap values supporting the differentiation of this family of RadA paralogs from both euryarchaeal RadB and the archaeal RadA proteins Figure 1reviewed in The aRadC family are differentiated Clock karser ks 203 инструкция the canonical archaeal recombinase RadA as, like RadB, they lack the NTD and are restricted to the core ATP binding domain.

This was comparable to Clock karser ks 203 инструкция rate observed for S. ATP hydrolysis by Sso was less efficient in the presence Clock karser ks 203 инструкция dsDNA and in the absence of DNA. ATP hydrolysis is not an inherent mechanistic requirement for the strand exchange process catalysed by RecA family members, but is thought to provide Clock karser ks 203 инструкция and stability and allow more extensive strand exchange reactions to proceed In contrast, RadB from P.

RadB lacks the NTD present in RadA but has the core ATPase domain of the RecA family RadB appears to lack the strand-exchange activity of RadA, and turns over ATP very slowly 18 ; Several crenarchaeal genomes encode multiple RadA paralogs, including three in S.

Drop sizes were 0. The initial hits were optimized using screens generated using an in-house stochastic optimization screen generator. Crystals were then frozen by rapid immersion in liquid nitrogen, and transferred to sample changer baskets Molecular Dimensions. Both crystals were screened at BM14UK Grenoble; ESRF using the sample changer, and a 2. Data were indexed and scaled with XDS and XSCALE 47 statistics are shown in Table 2 Clock karser ks 203 инструкция space group p Initial phases were determined by molecular replacement with Phaser 49 ; 50 as implemented in the CCP4 51 suite of programs, using the full length model RecA superfamily ATPase Pho from P.

However this motif is not present in the aRadC family Figure 5C. Each of these residues has been shown to be important for ssDNA binding in S. In summary, the structures of Sso and Pho highlight the key differences between the aRadC family and canonical RadA that can be related to their differing functions.

Therefore, the Sso protein was tested for ATP hydrolysis activity in the presence and absence of single- and double-stranded Clock karser ks 203 инструкция. The rate Clock karser ks 203 инструкция Ssomediated ATP hydrolysis was calculated to be approximately 0.

Both RadA and Sso have been reported as up-regulated in response to DNA damage by actinomycin D in S. The expression of all four genes remained relatively stable up to min Clock karser ks 203 инструкция UV treatment, in agreement with microarray data Clock karser ks 203 инструкция S. It is possible that protein levels are influenced at a post-translational stage, explaining the modest induction in RadA and Sto observed by Sheng and co-workers Our findings suggest that UV radiation and actinomycin D cause distinct transcriptional responses in S.

However, Clock karser ks 203 инструкция activity was observed for Sso alone. Furthermore, Sso inhibited the recombination activity of RadA Figure 4Cregardless of the order of addition.

In this study we investigated the expression levels and transcriptional response to UV damage for RadA and all three aRadC proteins from S. We cloned and expressed sso from S. Sso displays ssDNA-stimulated ATPase activity and binds ssDNA with nanomolar affinity. Although the protein can support the formation of small DNA heteroduplexes in an ATP-independent manner, it fails to catalyse the formation of D-loops, a key intermediate of homologous recombination, and inhibits RadA in these assays.

The hexameric quaternary structure of Pho is consistent with the known propensity of Clock karser ks 203 инструкция recombinases to adopt 6- 7- and 8-membered rings as well as a variety of helical forms The biochemical properties of Sso differentiate it from RadA.

However, no D-loops were formed, suggesting that under these conditions Sso Clock karser ks 203 инструкция not function as a mediator by displacing SSB to allow RadA-catalysed strand exchange. These data are in contrast to those of Sheng and co-workers, who suggested such a mediator function for the equivalent protein from Sulfolobus tokadaii D-loop formation assays for RadA and Sso The crystal structure of recombinant Sso was solved by molecular replacement using the structure deposited for protein Pho PDB code 2dr3, unpublished Figure 5A.

In contrast, SSB proteins bind tightly to ssDNA and are inhibitory to strand exchange reactions. Strand-exchange Activity of Sso and RadA. A more stringent test of recombinase activity is an assay for D-loop formation, Figure 4Awhich monitors the ability of a ssDNA molecule to invade a duplex DNA plasmid. Samples were taken at regular time intervals and deproteinised followed by agarose gel electrophoresis and phosphorimaging. Under these conditions, both E.

The archaea, although lacking a nucleus and bearing a superficial resemblance to bacteria, are more closely related to eukaryotes with respect to their informational processes, including DNA replication, recombination and repair 11transcription 12and translation Structural studies of HR proteins from archaea, including Rad50, Mre11 and RadA, have supplied a great deal of very useful information relevant to their eukaryal equivalents 14 ; Archaeal RadA is much more similar to Rad51 than to RecA, sharing the dsDNA binding N-terminal domain NTDand lacking the RecA-specific C-terminal domain CTD A second Rad51 paralog, RadB, has been described in some euryarchaea.

This model was refined using Refmac5 54 ; 55with manual readjustment using Coot TLS refinement was also used in the later rounds of the refinement process 57 ; Structure quality was checked using tools within Coot, and also by Molprobity The final model statistics are shown in Table 1.

The data, summarised in Table 1revealed that mRNA for radA was present at the highest levels under these conditions. The most highly transcribed paralog was sso, with transcript levels only 5-fold lower than those for radA, suggesting that it may be present in appreciable quantities in the cell. This is consistent with previous observations that Sso could be affinity purified from S.

Pure protein was analysed by electrospray mass spectrometry to confirm the identity and integrity of protein. Crystallization and Structure Solution. Crystallisation conditions were screened using a nano-drop crystallisation robot Cartesian Honeybee, Genomic Solutions as part of the Hamilton-Thermo Rhombix system, using commercially available sparse-matrix screens and one in-house screen.

These results were consistent with DNA-mediated assembly of the protein into a nucleoprotein complex as observed for other RecA family members including the ortholog from S.

The structure of Sso reveals a canonical RecA-type core fold, but there Clock karser ks 203 инструкция key differences in the DNA binding regions compared to RadA. The potential roles of RadA paralogs in archaea are discussed. Expression and UV inducibility of RadA and paralogs in S. Using quantitative one-step reverse transcriptase-PCR, we quantified the levels of mRNA transcripts encoding RadA and the three aRadC proteins in exponentially-growing S.

Firstly, Sso was assayed for heteroduplex formation Clock karser ks 203 инструкция a [32P]-radiolabelled nt DNA and an unlabelled duplex of 25 base pairs modified from reference 30 Figure 3A. Both RadA and Sso catalysed strand exchange at comparable rates Figure 3Band this activity was also observed when the two proteins were added consecutively, in either order, without any significant change in rate.

This type of interaction assay is susceptible to the generation of false positives when proteins have a DNA binding activity in common. When the possibility for DNA bridging is Clock karser ks 203 инструкция by the presence of ethidium bromide we have seen no evidence for a stable interaction between S.

These genes are all essential in mice, the proteins cooperate with Rad51 in strand-exchange reactions in vitro, and are required for damage-specific Rad51 repair foci in vivo 8 ; 9. However, there is no precise understanding of the molecular roles of the Rad51 paralogs, and the Rad51C-XRCC3 complex has also been implicated in the latter stages of the HR pathway 9.

There is also some similarity to the cyanobacterial circadian clock protein KaiC, the N-terminal domain of which is known to bind ATP and adopt a hexameric ring structure However, KaiC has a duplicated RecA domain and the partner protein KaiA is not present in any archaea, ruling out a role in circadian rhythm in the archaea. Phylogenetic analysis of archaeal RadA paralogs. These bioinformatics analyses reinforced the expectation aRadC proteins play a role in some aspect of DNA recombination in conjunction with RadA.

These data suggest that aRadC and RadB may have different functions in vivo, and this is supported by the observation that many euryarchaea including P. Protein interactions between eukaryotic recombination proteins are commonplace and may also be relevant in archaea.

Disruption of RecA function in bacteria, or Rad51 in yeast, is Clock karser ks 203 инструкция deleterious but not fatal to the cell, whereas in metazoa Rad51 is an essential protein 3. In contrast to bacteria where only a single RecA protein suffices, eukarya tend to encode a number of Rad51 paralogs in addition to Rad51 itself 5. There are seven RADlike genes in humans, comprising RAD51A, RAD51B, RAD51C, RAD51D, XRCC2, XRCC3 and the meiosis-specific DMC1 5. Five of these paralogs exist in two complexes in vivo: the BCDX2 complex RAD51B, RAD51C, RAD51D, and XRCC2 and the RAD51C-XRCC3 complex 6 ; 7.

The former binds ssDNA around fold more tightly than the latter. Although supporting limited ATP-independent strand exchange in vitro, Sso cannot catalyse D-loop formation, suggesting that it does not function as a recombinase in vivo. This is consistent with the known properties of P. Our results indicate that, in vitro, Sso cannot overcome the inhibitory effect of SSB-coated DNA on strand exchange by RadA.

Reactions were carried out in triplicate. The gene-specific primers used for amplification were as follows:.

Gene Cloning and Protein Expression. The sso gene from S. The N-terminal poly-histidine tag was cleaved by incubation with the Tobacco Etch Virus protease during Clock karser ks 203 инструкция purification. Mass spectrometry determined an intact mass of Sso is a DNA-dependent ATPase. A characteristic feature of the bacterial, archaeal and eukaryotic recombinases RecA, RadA and Rad51, respectively, is their ability to catalyse DNA-dependent ATP hydrolysis.

The final model contains protein residues, 98 water atoms, a pyrophosphate moiety, and four zinc atoms. There are small regions of Clock karser ks 203 инструкция between residues ssDNA binding loop 2 and residues 93 to The last 27 residues at the C-terminus were not visible in the electron density. There is a single non-prolyl cis peptide, serinewhich has been observed in other RecA superfamily members and appears to be a hallmark of the protein class reviewed in The Sso structure which contains a monomer in the asymmetric unit does not form any obviously biologically relevant quaternary structure by crystal packing.

These data suggest that Sso binds much more tightly to ssDNA than does RadA. The identification of Sso as one of a handful of proteins that can be affinity purified from cell extracts of S. Size exclusion chromatography using an analytical Superdex column GE Healthcare revealed that Sso was monomeric in solution in the absence of DNA, but formed large molecular weight complexes with predominant peaks at approximately and kDa when incubated with a 34mer oligonucleotide Figure 2C.

Sso is a good structural match to the core domain of RadA from S. The lack of these features probably corresponds to a fundamentally different role for the RadB and aRadC proteins, Clock karser ks 203 инструкция an inability to function as recombinases Clock karser ks 203 инструкция their own.

Sso binds tightly to ssDNA. The affinities of Sso for ssDNA and dsDNA were determined by fluorescence anisotropy experiments in which protein was titrated into a solution containing fluorescein-labelled single- or double-stranded DNA 45 nucleotides or base pairs, respectively.

It is also informative to compare the properties of aRadC and the euryarchaeal-specific RadB protein. Both bind tightly to ssDNA and inhibit RadA-mediated strand exchange in vitro. Their overall structures are very similar. However, RadB is expressed at a very low level in the cell Clock karser ks 203 инструкция does not turn over ATP Clock karser ks 203 инструкция vitro In contrast, Sso displays a robust ssDNA-dependent ATPase activity similar to that observed for RadA.

This does not rule out such a function in vivo, and reactions could potentially depend upon DNA helicases to help displace SSB, or on other proteins not present in the D-loop assays. Furthermore, our data suggest that Clock karser ks 203 инструкция can prevent RadA-mediated D-loop formation when incubated with ssDNA either before or after the addition of RadA.

The possible roles of the archaeal RadA paralogs in vivo are discussed. Keywords: Clock karser ks 203 инструкция, Recombinase, RadA, Homologous Recombination, Strand Exchange. The RecA protein family, comprising Rad51 and its paralogs in eukarya, RadA in archaea and RecA in bacteria, is one of the few universally conserved Clock karser ks 203 инструкция repair proteins. RecA family members are DNA recombinases catalysing strand-exchange Clock karser ks 203 инструкция that are central to homologous recombination HR and double-strand break repair DSBR 1.

The hexameric quaternary structure of Pho is shown in Figure 5Bin which one subunit is overlaid with the Sso structure. Contained on this loop are several residues involved in stabilizing the Pho interface, including Glu, Glu and Leu, and Asp, which forms a salt bridge with Arg Pho contains ADP in the active site, in a position equivalent to pyrophosphate in the Sso structure.

All archaea have at least one and as Clock karser ks 203 инструкция as four RadA paralogs, but their function remains unclear. Here we show the three RadA paralogs encoded by the Sulfolobus solfataricus genome are expressed under normal growth conditions, and are Clock karser ks 203 инструкция UV-inducible. We demonstrate that one of these proteins, Sso, which is representative of the large aRadC sub-family of archaeal RadA paralogs, functions as an ATPase that binds tightly to ssDNA.

ATPase activity and DNA binding affinity of Sso Strand exchange and D-loop formation by Sso To assess the ability of Sso to catalyse strand exchange, two assays were performed.

Like RadA, none were induced by UV radiation. This seems to be a characteristic of DNA repair proteins in hyperthermophiles, where harsh environments require constitutive DNA repair We have gone on to characterise one of these proteins, Sso, which is representative of the large family of aRadC paralogs The structure of Sso is likely Clock karser ks 203 инструкция be representative Clock karser ks 203 инструкция the aRadC family in general, and is closely related to the unpublished structure of Pho, an aRadC family member from P.

Further biochemical and genetic characterisation of the other archaeal RadA paralogs will be required to delineate their role in Homologous Recombination. Preparation of DNA substrates. Substrates were subsequently purified by native acrylamide gel electrophoresis. Harvested cells were lysed by sonication in lysis buffer Clock karser ks 203 инструкция mM Tris-HCl [pH 7. The protein was bound to a nickel-chelating column HiTrap 5 ml Chelating HP, GE Healthcare equilibrated with column buffer 20 mM Tris-HCl [pH 7.

See other articles in PMC that cite the published article. DNA recombinases RecA in bacteria, Rad51 in eukarya and RadA in archaea catalyse strand-exchange between homologous DNA molecules, the central reaction of homologous recombination, and are among the most conserved DNA repair proteins known. In bacteria, RecA is the sole protein responsible for this reaction, whereas, in eukaryotes, there are several RAD51 paralogs that cooperate to catalyse strand exchange.

They bind single-stranded DNA ssDNAforming a nucleoprotein filament that can invade duplex DNA with a cognate sequence, leading to the formation of recombination intermediates such as heteroduplexes, D-loops and Holliday junctions 2.