Structure and cellular dynamics of Deinococcus radiodurans single-stranded DNA (ssDNA)-binding protein (SSB)-DNA complexes

The single-stranded DNA (ssDNA)-binding protein from the radiation-resistant bacterium Deinococcus radiodurans (DrSSB) functions as a homodimer in which each monomer contains two oligonucleotide-binding (OB) domains. This arrangement is exceedingly rare among bacterial SSBs, which typically form homotetramers of single-OB domain subunits. To better understand how this unusual structure influences the DNA binding and biological functions of DrSSB in D. radiodurans radiation resistance, we have examined the structure of DrSSB in complex with ssDNA and the DNA damage-dependent cellular dynamics of DrSSB. The x-ray crystal structure of the DrSSB-ssDNA complex shows that ssDNA binds to surfaces of DrSSB that are analogous to those mapped in homotetrameric SSBs, although there are distinct contacts in DrSSB that mediate species-specific ssDNA binding. Observations by electron microscopy reveal two salt-dependent ssDNA-binding modes for DrSSB that strongly resemble those of the homotetrameric Escherichia coli SSB, further supporting a shared overall DNA binding mechanism between the two classes of bacterial SSBs. In vivo, DrSSB levels are heavily induced following exposure to ionizing radiation. This accumulation is accompanied by dramatic time-dependent DrSSB cellular dynamics in which a single nucleoid-centric focus of DrSSB is observed within 1 h of irradiation but is dispersed by 3 h after irradiation. These kinetics parallel those of D. radiodurans postirradiation genome reconstitution, suggesting that DrSSB dynamics could play important organizational roles in DNA repair.     

Novel thermostable single-stranded DNA-binding protein (SSB) from Deinococcus geothermalis

To study the biochemical properties of single-stranded DNA-binding (SSB) protein from Deinococcus geothermalis (DgeSSB), we have cloned the ssb gene obtained by PCR and developed an overexpression system. The gene consists of an open reading frame of 900 nucleotides encoding a protein of 300 amino acids with a calculated molecular weight of 32.45 kDa. The amino acid sequence exhibits 43, 44 and 75% identity with Thermus aquaticus, Thermus thermophilus and Deinococcus radiodurans SSBs, respectively. We show that DgeSSB is similar to Thermus/Deinococcus SSB in its biochemical properties. DgeSSB includes two oligonucleotide/oligosaccharide-binding folds per monomer and functions as a homodimer. In fluorescence titrations with poly(dT), DgeSSB bound about 30 nt independent of the salt concentration, and the fluorescence was quenched by about 65%. In a complementation assay in Escherichia coli, DgeSSB took over the in vivo function of EcoSSB. DgeSSB is thermostable with half-lives of 50 min at 70°C and 5 min at 90°C. Hence, DgeSSB offers an attractive alternative for TaqSSB and TthSSB in their applications for molecular biology methods and for analytical purposes.     

Identification and properties of the Deinococcus grandis and Deinococcus proteolyticus single-stranded DNA binding proteins (SSB)

To study the biochemical properties of SSB's from Deinococcus grandis (DgrSSB) and Deinococcus proteolyticus (DprSSB), we have cloned the ssb genes obtained by PCR and have developed Escherichia coli overexpression systems. The genes consist of an open reading frame of 891 (DgrSSB) and 876 (DprSSB) nucleotides encoding proteins of 296 and 291 amino acids with a calculated molecular mass of 32.29 and 31.33 kDa, respectively. The amino-acid sequence of DgrSSB exhibits 45%, 44% and 82% identity and the amino-acid sequence of DprSSB reveals 43%, 43% and 69% identity with Thermus aquaticus (TaqSSB), Thermus thermophilus (TthSSB) and Deinococcus radiodurans SSBs, respectively. We show that DgrSSB and DprSSB are similar to Thermus/Deinococcus SSBs in their biochemical properties. They are functional as homodimers, with each monomer encoding two single-stranded DNA binding domains (OB-folds). In fluorescence titrations with poly(dT), both proteins bind single-stranded DNA with a binding site size of about 33 nt per homodimer. In a complementation assay in E. coli, DgrSSB and DprSSB took over the in vivo function of EcoSSB. Thermostability with half-lives of about 1 min at 65-67.5 degrees C make DgrSSB and DprSSB similar to the known SSB of Deinococcus radiodurans (DraSSB).     

DdrB stimulates single-stranded DNA annealing and facilitates RecA-independent DNA repair in Deinococcus radiodurans

The bacterium Deinococcus radiodurans can survive extremely high exposure to ionizing radiation. The repair mechanisms involved in this extraordinary ability are still being investigated. ddrB is one gene that is highly up-regulated after irradiation, and it has been proposed to be involved in RecA-independent repair in D. radiodurans. Here we cloned, expressed and characterized ddrB in order to define its roles in the radioresistance of D. radiodurans. DdrB preferentially binds to single-stranded DNA. Moreover, it interacts directly with single-stranded binding protein of D. radiodurans DrSSB, and stimulates single-stranded DNA annealing even in the presence of DrSSB. The post-irradiation DNA repair kinetics of a ddrB/recA double mutant were compared to ddrB and recA single mutants by pulsed-field gel electrophoresis (PFGE). DNA fragment rejoining in the ddrB/recA double mutant is severely compromised, suggesting that DdrB-mediated single-stranded annealing plays a critical role in the RecA-independent DNA repair of D. radiodurans.     

DNA helicase activity of the RecD protein from Deinococcus radiodurans

The bacterium Deinococcus radiodurans is extremely resistant to high levels of DNA-damaging agents, including gamma rays and ultraviolet light that can lead to double-stranded DNA breaks. Surprisingly, the organism does not appear to have a RecBCD enzyme, an enzyme that is critical for double-strand break repair in many other bacteria. The D. radiodurans genome does encode a protein whose closest characterized homologues are RecD subunits of RecBCD enzymes in other bacteria. We have purified this novel D. radiodurans RecD protein and characterized its biochemical activities. The D. radiodurans RecD protein is a DNA helicase that unwinds short (20 base pairs) DNA duplexes with either a 5'-single-stranded tail or a forked end, but not blunt-ended or 3'-tailed duplexes. Duplexes with 10-12 nucleotide (nt) 5'-tails are good unwinding substrates and are bound tightly, while DNA with shorter tails (4-8 nt) are poor unwinding substrates and are bound much less tightly. The RecD protein is much less efficient at unwinding slightly longer substrates (52 or 76 base pairs, with 12 nt 5'-tails). Unwinding of the longer substrates is stimulated somewhat (4-5-fold) by the single-stranded DNA-binding protein from D. radiodurans. These results show that the D. radiodurans RecD protein is a DNA helicase with 5'-3' polarity and low processivity.     

Interaction of double-stranded DNA with polymerized PprA protein from Deinococcus radiodurans

Pleiotropic protein promoting DNA repair A (PprA) is a key protein that facilitates the extreme radioresistance of Deinococcus radiodurans. To clarify the role of PprA in the radioresistance mechanism, the interaction between recombinant PprA expressed in Escherichia coli with several double-stranded DNAs (i.e., super coiled, linear, or nicked circular dsDNA) was investigated. In a gel-shift assay, the band shift of supercoiled pUC19 DNA caused by the binding of PprA showed a bimodal distribution, which was promoted by the addition of 1 mM Mg, Ca, or Sr ions. The dissociation constant of the PprA-supercoiled pUC19 DNA complex, calculated from the relative portions of shifted bands, was 0.6 μM with Hill coefficient of 3.3 in the presence of 1 mM Mg acetate. This indicates that at least 281 PprA molecules are required to saturate a supercoiled pUC19 DNA, which is consistent with the number (280) of bound PprA molecules estimated by the UV absorption of the PprA–pUC19 complex purified by gel filtration. This saturation also suggests linear polymerization of PprA along the dsDNA. On the other hand, the bands of linear dsDNA and nicked circular dsDNA that eventually formed PprA complexes did not saturate, but created larger molecular complexes when the PprA concentration was >1.3 μM. This result implies that DNA-bound PprA aids association of the termini of damaged DNAs, which is regulated by the concentration of PprA. These findings are important for the understanding of the mechanism underlying effective DNA repair involving PprA.     

SSB protein limits RecOR binding onto single-stranded DNA

The RecO and RecR proteins form a complex that promotes the nucleation of RecA protein filaments onto SSB protein-coated single-stranded DNA (ssDNA). However, even when RecO and RecR proteins are provided at optimal concentrations, the loading of RecA protein is surprisingly slow, typically proceeding with a lag of 10 min or more. The rate-limiting step in RecOR-promoted RecA nucleation is the binding of RecOR protein to ssDNA, which is inhibited by SSB protein despite the documented interaction between RecO and SSB. Full activity of RecOR is seen only when RecOR is preincubated with ssDNA prior to the addition of SSB. The slow binding of RecOR to SSB-coated ssDNA involves the C terminus of SSB. When an SSB variant that lacks the C-terminal 8 amino acids is used, the capacity of RecOR to facilitate RecA loading onto the ssDNA is largely abolished. The results are used in an expanded model for RecOR action.     

A novel structure of DNA repair protein RecO from Deinococcus radiodurans

Recovery of arrested replication requires coordinated action of DNA repair, replication, and recombination machineries. Bacterial RecO protein is a member of RecF recombination repair pathway important for replication recovery. RecO possesses two distinct activities in vitro, closely resembling those of eukaryotic protein Rad52: DNA annealing and RecA-mediated DNA recombination. Here we present the crystal structure of the RecO protein from the extremely radiation resistant bacteria Deinococcus radiodurans (DrRecO) and characterize its DNA binding and strand annealing properties. The RecO structure is totally different from the Rad52 structure. DrRecO is comprised of three structural domains: an N-terminal domain which adopts an OB-fold, a novel alpha-helical domain, and an unusual zinc-binding domain. Sequence alignments suggest that the multidomain architecture is conserved between RecO proteins from other bacterial species and is suitable to elucidate sites of protein-protein and DNA-protein interactions necessary for RecO functions during the replication recovery and DNA repair.     

Analysis of interaction between DNA and Deinococcus radiodurans PprA protein by atomic force microscopy

A DNA repair-promoting protein, PprA, was isolated from a radiation resistant bacterium, Deinococcus radiodurans [I. Narumi, K. Sato, S. Cui, T. Funayama, S. Kitayama, H. Watanabe, PprA: a novel protein from Deinococcus radiodurans that stimulates DNA ligation, Mol. Microbiol. 54 (2004) 278-285]. Despite several studies, however, the function of PprA is not still clear. We used atomic force microscopy (AFM) to elucidate the role of this protein in the DNA repair pathway. In the present study, interaction between the linear DNA and PprA protein was imaged and analyzed by AFM without any fixation or staining. Though both end-bound and internally bound PprA was observed, the affinity of the end-bound protein was greater considering the proportion of features of binding analyzed by AFM. In some conditions, looping forms of the DNA-PprA complex were observed. Gel filtration high performance liquid chromatography (HPLC) was also conducted to estimate the molecular weight of this protein. The result of the HPLC analysis suggested that PprA formed multimers in buffer solution without DNA.     

PprA: a novel protein from Deinococcus radiodurans that stimulates DNA ligation

The extraordinary radiation resistance of Deinococcus radiodurans results from the efficient capacity of the bacterium to repair DNA double-strand breaks. By analysing the DNA damage repair-deficient mutant, KH311, a unique radiation-inducible gene (designated pprA) responsible for loss of radiation resistance was identified. Investigations in vitro showed that the gene product of pprA (PprA) preferentially bound to double-stranded DNA carrying strand breaks, inhibited Escherichia coli exonuclease III activity, and stimulated the DNA end-joining reaction catalysed by ATP-dependent and NAD-dependent DNA ligases. These results suggest that D. radiodurans has a radiation-induced non-homologous end-joining repair mechanism in which PprA plays a critical role.     

Kinetics of DNA Unwinding by the RecD2 Helicase from Deinococcus radiodurans

RecD2 from Deinococcus radiodurans is a superfamily 1 DNA helicase that is homologous to the Escherichia coli RecD protein but functions outside the context of RecBCD enzyme. We report here on the kinetics of DNA unwinding by RecD2 under single and multiple turnover conditions. There is little unwinding of 20-bp substrates by preformed RecD2-dsDNA complexes when excess ssDNA is present to trap enzyme molecules not bound to the substrate. A shorter 12-bp substrate is unwound rapidly under single turnover conditions. The 12-bp unwinding reaction could be simulated with a mechanism in which the DNA is unwound in two kinetic steps with rate constant of k_unw = 5.5 s⁻¹ and a dissociation step from partially unwound DNA of k_off = 1.9 s⁻¹. These results indicate a kinetic step size of about 3–4 bp, unwinding rate of about 15–20 bp/s, and low processivity (p = 0.74). The reaction time courses with 20-bp substrates, determined under multiple turnover conditions, could be simulated with a four-step mechanism and rate constant values very similar to those for the 12-bp substrate. The results indicate that the faster unwinding of a DNA substrate with a forked end versus only a 5′-terminal single-stranded extension can be accounted for by a difference in the rate of enzyme binding to the DNA substrates. Analysis of reactions done with different RecD2 concentrations indicates that the enzyme forms an inactive dimer or other oligomer at high enzyme concentrations. RecD2 oligomers can be detected by glutaraldehyde cross-linking but not by size exclusion chromatography.     

Histone-like protein HU from Deinococcus radiodurans binds preferentially to four-way DNA junctions

The histone-like protein HU from Escherichia coli is involved in DNA compaction and in processes such as DNA repair and recombination. Its participation in these events is reflected in its ability to bend DNA and in its preferred binding to DNA junctions and DNA with single-strand breaks. Deinococcus radiodurans is unique in its ability to reconstitute its genome from double strand breaks incurred after exposure to ionizing radiation. Using electrophoretic mobility shift assays (EMSA), we show that D.radiodurans HU (DrHU) binds preferentially only to DNA junctions, with half-maximal saturation of 18 nM. In distinct contrast to E.coli HU, DrHU does not exhibit a marked preference for DNA with nicks or gaps compared to perfect duplex DNA, nor is it able to mediate circularization of linear duplex DNA. These unexpected properties identify DrHU as the first member of the HU protein family not to serve an architectural role and point to its potential participation in DNA recombination events. Our data also point to a mechanism whereby differential target site selection by HU proteins is achieved and suggest that the substrate specificity of HU proteins should be expected to vary as a consequence of their individual capacity for inducing the required DNA bend.     

Investigating Deinococcus radiodurans RecA protein filament formation on double-stranded DNA by a real-time single-molecule approach

With the aid of an efficient, precise, and almost error-free DNA repair system, Deinococcus radiodurans can survive hundreds of double-strand breaks inflicted by high doses of irradiation or desiccation. RecA of D. radiodurans (DrRecA) plays a central role both in the early phase of repair by an extended synthesis-dependent strand annealing process and in the later more general homologous recombination phase. Both roles likely require DrRecA filament formation on duplex DNA. We have developed single-molecule tethered particle motion experiments to study the assembly dynamics of RecA proteins on individual duplex DNA molecules by observing changes in DNA tether length resulting from RecA binding. We demonstrate that DrRecA nucleation on double-stranded DNA is much faster than that of Escherichia coli RecA protein (EcRecA), but the extension is slower. This combination of attributes would tend to increase the number and decrease the length of DrRecA filaments relative to those of EcRecA, a feature that may reflect the requirement to repair hundreds of genomic double-strand breaks concurrently in irradiated Deinococcus cells.     

DNA repair in the extremely radioresistant bacterium Deinococcus radiodurans

Deinococcus radiodurans and other members of the same genus share extraordinary resistance to the lethal and mutagenic effects of ionizing and u.v. radiation and to many other agents that damage DNA. While it is known that this resistance is due to exceedingly efficient DNA repair, the molecular mechanisms responsible remain poorly understood. Following very high exposures to u.v. irradiation (e.g. 500 Jm⁻², which is non-lethal to D. radiodurans), this organism carries out extremely efficient excision repair accomplished by two separate nucleotide excision repair pathways acting simultaneously. One pathway requires the uvrA gene and appears similar to the UvrABC excinuclease pathway defined in Escherichia coli. The other excision repair pathway is specific for u.v. dimeric photoproducts, but is not mediated by a pyrimidine dimer DNA glycosylase. Instead, it is initiated by a second bona fide endonuclease that may recognize both pyrimidine dimers and pyrimidine-(6–4)pyrimidones. After high doses of ionizing-radiation (e.g. 1.5Mrad), D. radiodurans can mend >100 double-strand breaks (dsb) per chromosome without lethality or mutagenesis. Both dsb mending and survival are recA-dependent, indicating that efficient dsb mending proceeds via homologous recombination. D. radiodurans contains multiple chromosomes per cell, and it is proposed that dsb mending requires extensive recombination amongst these chromosomes, a novel phenomenon in bacteria. Thus, D. radiodurans may serve as an easily accessible model system for the double-strand-break-initiated interchromosomal recombination that occurs in eukaryotic cells during mitosis and meiosis.     

A mechanism for single-stranded DNA-binding protein (SSB) displacement from single-stranded DNA upon SSB-RecO interaction

Displacement of single-stranded DNA (ssDNA)-binding protein (SSB) from ssDNA is necessary for filament formation of RecA on ssDNA to initiate homologous recombination. The interaction between RecO and SSB is considered to be important for SSB displacement; however, the interaction has not been characterized at the atomic level. In this study, to clarify the mechanism underlying SSB displacement from ssDNA upon RecO binding, we examined the interaction between Thermus thermophilus RecO and cognate SSB by NMR analysis. We found that SSB interacts with the C-terminal positively charged region of RecO. Based on this result, we constructed some RecO mutants. The R127A mutant had considerably decreased binding affinity for SSB and could not anneal SSB-coated ssDNAs. Further, the mutant in the RecOR complex prevented the recovery of ssDNA-dependent ATPase activity of RecA from inhibition by SSB. These results indicated that the region surrounding Arg-127 is the binding site of SSB. We also performed NMR analysis using the C-terminal peptide of SSB and found that the acidic region of SSB is involved in the interaction with RecO, as seen in other protein-SSB interactions. Taken together with the findings of previous studies, we propose a model for SSB displacement from ssDNA where the acidic C-terminal region of SSB weakens the ssDNA binding affinity of SSB when the dynamics of the C-terminal region are suppressed by interactions with other proteins, including RecO.     

Single molecule analysis of Thermus thermophilus SSB protein dynamics on single-stranded DNA

Single-stranded (ss) DNA binding (SSB) proteins play central roles in DNA replication, recombination and repair in all organisms. We previously showed that Escherichia coli (Eco) SSB, a homotetrameric bacterial SSB, undergoes not only rapid ssDNA-binding mode transitions but also one-dimensional diffusion (or migration) while remaining bound to ssDNA. Whereas the majority of bacterial SSB family members function as homotetramers, dimeric SSB proteins were recently discovered in a distinct bacterial lineage of extremophiles, the Thermus–Deinococcus group. Here we show, using single-molecule fluorescence resonance energy transfer (FRET), that homodimeric bacterial SSB from Thermus thermophilus (Tth) is able to diffuse spontaneously along ssDNA over a wide range of salt concentrations (20–500 mM NaCl), and that TthSSB diffusion can help transiently melt the DNA hairpin structures. Furthermore, we show that two TthSSB molecules undergo transitions among different DNA-binding modes while remaining bound to ssDNA. Our results extend our previous observations on homotetrameric SSBs to homodimeric SSBs, indicating that the dynamic features may be shared among different types of SSB proteins. These dynamic features of SSBs may facilitate SSB redistribution and removal on/from ssDNA, and help recruit other SSB-interacting proteins onto ssDNA for subsequent DNA processing in DNA replication, recombination and repair.     

Substrate specificity of Deinococcus radiodurans Fpg protein.

A DNA repair enzyme has recently been isolated from the ionizing radiation-resistant bacterium Deinococcus radiodurans [Bauche, C., and Laval, J. (1999) J. Bacteriol. 181, 262-269]. This enzyme is a homologue of the Fpg protein of Escherichia coli. We investigated the substrate specificity of this enzyme for products of oxidative DNA base damage using gas chromatography/isotope-dilution mass spectrometry and DNA substrates, which were either gamma-irradiated or treated with H(2)O(2)/Fe(III)-EDTA/ascorbic acid. Excision of purine lesions 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), 4,6-diamino-5-formamidopyrimidine (FapyAde), and 8-hydroxyguanine (8-OH-Gua) was observed among 17 lesions detected in damaged DNA substrates. The extent of excision was determined as a function of enzyme concentration, time, and substrate concentration. FapyGua and FapyAde were excised with similar specificities from three DNA substrates, whereas 8-OH-Gua was the least preferred lesion. The results show that D. radiodurans Fpg protein and its homologue E. coli Fpg protein excise the same modified DNA bases, but the excision rates of these enzymes are significantly different. Formamidopyrimidines are preferred substrates of D. radiodurans Fpg protein over 8-OH-Gua, whereas E. coli Fpg protein excises these three lesions with similar efficiencies from various DNA substrates. Substrate specificities of these enzymes were also compared with that of Saccharomyces cerevisiae Ogg1 protein, which excises FapyGua and 8-OH-Gua, but not FapyAde.     

Deinococcus radiodurans DNA increases the radiation resistance of Escherichia coli

A genomic DNA library of Deinococcus radiodurans DNA has been prepared using the plasmid vector pBR322. The recombinant plasmid was used to transform a more radiation-sensitive organism, Escherichia coli RR1. Following selection of transformed organisms by their ability to grow on ampicillin, radiation-resistant organisms were selected by irradiation with 137Cs gamma radiation. Increased radiation resistance correlates with the presence of a 3-kb fragment of DNA in these cells which is derived from D. radiodurans.     

RecA protein assures fidelity of DNA repair and genome stability in Deinococcus radiodurans

Deinococcus radiodurans is one of the most radiation-resistant organisms known. It can repair hundreds of radiation-induced double-strand DNA breaks without loss of viability. Genome reassembly in heavily irradiated D. radiodurans is considered to be an error-free process since no genome rearrangements were detected after post-irradiation repair. Here, we describe for the first time conditions that frequently cause erroneous chromosomal assemblies. Gross chromosomal rearrangements have been detected in recA mutant cells that survived exposure to 5 kGy γ-radiation. The recA mutants are prone also to spontaneous DNA rearrangements during normal exponential growth. Some insertion sequences have been identified as dispersed genomic homology blocks that can mediate DNA rearrangements. Whereas the wild-type D. radiodurans appears to repair accurately its genome shattered by 5 kGy γ-radiation, extremely high γ-doses, e.g., 25 kGy, produce frequent genome rearrangements among survivors. Our results show that the RecA protein is quintessential for the fidelity of repair of both spontaneous and γ-radiation-induced DNA breaks and, consequently, for genome stability in D. radiodurans. The mechanisms of decreased genome stability in the absence of RecA are discussed.