A possible interaction of single-strand binding protein and RecA protein during post-ultraviolet DNA synthesis

The mechanism of DNA replication in ultraviolet (UV)-irradiated Escherichia coli is proposed. Immediately after UV exposure, the replisome aided by single-strand DNA-binding protein (SSB) can proceed past UV-induced pyrimidine dimers without insertion of nucleotides. Polymerisation eventually resumes somewhere downstream of the dimer sites. Due to the limited supply of SSB, only a few dimers can be bypassed in this way. Nevertheless, this early DNA synthesis is of great biological importance because it generates single-stranded DNA regions. Single-stranded DNA can bind and activate RecA protein, thus leading to induction of the SOS response. During the SOS response, the cellular level of RecA protein increases dramatically. Due to the simultaneous increase in the concentration of ATP, RecA protein achieves the high-affinity state for single-stranded DNA. Therefore it is able to displace DNA-bound SSB. The cycling of SSB on and off DNA enables the replisome to bypass a large number of dimers at late post-UV times. During this late replication, the stoichiometric amounts of RecA protein needed for recombination are involved in the process of postreplication repair.     

The E.coli RuvAB proteins branch migrate Holliday junctions through heterologous DNA sequences in a reaction facilitated by SSB.

During genetic recombination a heteroduplex joint is formed between two homologous DNA molecules. The heteroduplex joint plays an important role in recombination since it accommodates sequence heterogeneities (mismatches, insertions or deletions) that lead to genetic variation. Two Escherichia coli proteins, RuvA and RuvB, promote the formation of heteroduplex DNA by catalysing the branch migration of crossovers, or Holliday junctions, which link recombining chromosomes. We show that RuvA and RuvB can promote branch migration through 1800 bp of heterologous DNA, in a reaction facilitated by the presence of E.coli single-stranded DNA binding (SSB) protein. Reaction intermediates, containing unpaired heteroduplex regions bound by SSB, were directly visualized by electron microscopy. In the absence of SSB, or when SSB was replaced by a single-strand binding protein from bacteriophage T4 (gene 32 protein), only limited heterologous branch migration was observed. These results show that the RuvAB proteins, which are induced as part of the SOS response to DNA damage, allow genetic recombination and the recombinational repair of DNA to occur in the presence of extensive lengths of heterology.     

Mechanism of RecO recruitment to DNA by single-stranded DNA binding protein

RecO is a recombination mediator protein (RMP) important for homologous recombination, replication repair and DNA annealing in bacteria. In all pathways, the single-stranded (ss) DNA binding protein, SSB, plays an inhibitory role by protecting ssDNA from annealing and recombinase binding. Conversely, SSB may stimulate each reaction through direct interaction with RecO. We present a crystal structure of Escherichia coli RecO bound to the conserved SSB C-terminus (SSB-Ct). SSB-Ct binds the hydrophobic pocket of RecO in a conformation similar to that observed in the ExoI/SSB-Ct complex. Hydrophobic interactions facilitate binding of SSB-Ct to RecO and RecO/RecR complex in both low and moderate ionic strength solutions. In contrast, RecO interaction with DNA is inhibited by an elevated salt concentration. The SSB mutant lacking SSB-Ct also inhibits RecO-mediated DNA annealing activity in a salt-dependent manner. Neither RecO nor RecOR dissociates SSB from ssDNA. Therefore, in E. coli, SSB recruits RMPs to ssDNA through SSB-Ct, and RMPs are likely to alter the conformation of SSB-bound ssDNA without SSB dissociation to initiate annealing or recombination. Intriguingly, Deinococcus radiodurans RecO does not bind SSB-Ct and weakly interacts with the peptide in the presence of RecR, suggesting the diverse mechanisms of DNA repair pathways mediated by RecO in different organisms.     

Analysis of the SOS inducing signal in Bacillus subtilis using Escherichia coli LexA as a probe.

We analyzed the Bacillus subtilis SOS response using Escherichia coli LexA protein as a probe to measure the kinetics of SOS activation and DNA repair in wild-type and DNA repair-deficient strains. By examining the effects of DNA-damaging agents that produce the SOS inducing signal in E. coli by three distinct pathways, we obtained evidence that the nature of the SOS inducing signal has been conserved in B. subtilis. In particular, we used the B. subtilis DNA polymerase III inhibitor, 6-(p-hydroxyphenylazo)-uracil, to show that DNA replication is required to generate the SOS inducing signal following UV irradiation. We also present evidence that single-stranded gaps, generated by excision repair, serve as part of the UV inducing signal. By assaying the SOS response in B. subtilis dinA, dinB, and dinC mutants, we identified distinct deficiencies in SOS activation and DNA repair that suggest roles for the corresponding gene products in the SOS response.     

Lesion bypass by the Escherichia coli DNA polymerase V requires assembly of a RecA nucleoprotein filament

Translesion replication is carried out in Escherichia coli by the SOS-inducible DNA polymerase V (UmuC), an error-prone polymerase, which is specialized for replicating through lesions in DNA, leading to the formation of mutations. Lesion bypass by pol V requires the SOS-regulated proteins UmuD' and RecA and the single-strand DNA-binding protein (SSB). Using an in vitro assay system for translesion replication based on a gapped plasmid carrying a site-specific synthetic abasic site, we show that the assembly of a RecA nucleoprotein filament is required for lesion bypass by pol V. This is based on the reaction requirements for stoichiometric amounts of RecA and for single-stranded gaps longer than 100 nucleotides and on direct visualization of RecA-DNA filaments by electron microscopy. SSB is likely to facilitate the assembly of the RecA nucleoprotein filament; however, it has at least one additional role in lesion bypass. ATPgammaS, which is known to strongly increase binding of RecA to DNA, caused a drastic inhibition of pol V activity. Lesion bypass does not require stoichiometric binding of UmuD' along RecA filaments. In summary, the RecA nucleoprotein filament, previously known to be required for SOS induction and homologous recombination, is also a critical intermediate in translesion replication.     

Limited proteolysis studies on the Escherichia coli single-stranded DNA binding protein. Evidence for a functionally homologous domain in both the Escherichia coli and T4 DNA binding proteins

Limited proteolysis can be used to remove either 42 or 62 amino acids at the COOH terminus of the 18,873-dalton Escherichia coli single-stranded DNA binding protein (SSB). Since poly(dT), but not d(pT)16, increases the rate of this reaction, it appears that cooperative SSB binding to single-stranded DNA (ssDNA) is associated with a conformational change that increases the exposure of the COOH terminus to proteolysis. As a result of this DNA-induced conformational change, we presume that the COOH-terminal region of SSB will become more accessible for interacting with other proteins that utilize the SSB:ssDNA complex as a substrate and that are involved in E. coli DNA replication, repair, and recombination. Removal of this COOH-terminal domain from SSB results in a stronger helix-destabilizing protein which suggests this region may be important for controlling the ability of SSB to denature double-stranded DNA. Since similar results have previously been reported for the bacteriophage T4 gene 32 protein (Williams, K.R., and Konigsberg, W. (1978) J. Biol. Chem. 253, 2463-2470; Hosoda, J., and Moise, H. (1978) J. Biol. Chem. 253, 7547-7555), the acidic, COOH-terminal domains of these two single-stranded DNA binding proteins may be functionally homologous. Preliminary evidence is cited that suggests other prokaryotic and eukaryotic DNA binding proteins may contain similar functional domains essential for controlling their ability to invade double helical DNA.     

SSB蛋白与ssDNA相互作用的动力学和可视化研究

研究大肠杆菌单链结合蛋白(single-stranded DNA-binding protein,SSB)与单链DNA(single-stranded DNA,ssDNA)的相互作用对于了解其在DNA复制、重组和修复中的作用是非常重要的。通过表面等离子共振技术(surface plasmon resonance,SPR)得到了在有、无镁离子的情况下,SSB与ssDNA两者的平衡解离常数(equilibrium dissociation constant,KD)分别为9.67×10-7M和4.79×10-7M,阐明了镁离子对于两者作用形式的影响。利用原子力显微镜技术分别观察SSB蛋白、ssDNA和SSB-ssDNA复合物的成像,为下一步研究SSB在DNA代谢中作用模式的单分子可视化奠定了基础。     

Initiation of genetic recombination and recombination-dependent replication

Recombination initiates at double-stranded DNA breaks and at single-stranded DNA gaps. These DNA strand discontinuities can arise from DNA-damaging agents and from normal DNA replication when the DNA polymerase encounters an imperfection in the DNA template or another protein. The machinery of homologous recombination acts at these breaks and gaps to promote the events that result in gene recombination, as well as the reattachment of detached replication arms and the resumption of DNA replication. In Escherichia coli, these events require collaboration (RecA, RecBCD, RecFOR, RecQ, RuvABC and SSB proteins) and DNA replication (PriABC proteins and the DNA polymerases). The initial steps common to these recombination and recombination-dependent replication processes are reviewed.     

ssb Gene Duplication Restores the Viability of ΔholC and ΔholD Escherichia coli Mutants

The HolC-HolD (χψ) complex is part of the DNA polymerase III holoenzyme (Pol III HE) clamp-loader. Several lines of evidence indicate that both leading- and lagging-strand synthesis are affected in the absence of this complex. The Escherichia coli ΔholD mutant grows poorly and suppressor mutations that restore growth appear spontaneously. Here we show that duplication of the ssb gene, encoding the single-stranded DNA binding protein (SSB), restores ΔholD mutant growth at all temperatures on both minimal and rich medium. RecFOR-dependent SOS induction, previously shown to occur in the ΔholD mutant, is unaffected by ssb gene duplication, suggesting that lagging-strand synthesis remains perturbed. The C-terminal SSB disordered tail, which interacts with several E. coli repair, recombination and replication proteins, must be intact in both copies of the gene in order to restore normal growth. This suggests that SSB-mediated ΔholD suppression involves interaction with one or more partner proteins. ssb gene duplication also suppresses ΔholC single mutant and ΔholC ΔholD double mutant growth defects, indicating that it bypasses the need for the entire χψ complex. We propose that doubling the amount of SSB stabilizes HolCD-less Pol III HE DNA binding through interactions between SSB and a replisome component, possibly DnaE. Given that SSB binds DNA in vitro via different binding modes depending on experimental conditions, including SSB protein concentration and SSB interactions with partner proteins, our results support the idea that controlling the balance between SSB binding modes is critical for DNA Pol III HE stability in vivo, with important implications for DNA replication and genome stability.     

Biophysical analysis of Thermus aquaticus single-stranded DNA binding protein

Due to their involvement in processes such as DNA replication, repair, and recombination, bacterial single-stranded DNA binding (SSB) proteins are essential for the survival of the bacterial cell. Whereas most bacterial SSB proteins form homotetramers in solution, dimeric SSB proteins were recently discovered in the Thermus/Deinococcus group. In this work we characterize the biophysical properties of the SSB protein from Thermus aquaticus (TaqSSB), which is structurally quite similar to the tetrameric SSB protein from Escherichia coli (EcoSSB). The binding of TaqSSB and EcoSSB to single-stranded nucleic acids was found to be very similar in affinity and kinetics. Mediated by its highly conserved C-terminal region, TaqSSB interacts with the χ-subunit of E. coli DNA polymerase III with an affinity that is similar to that of EcoSSB. Using analytical ultracentrifugation, we show that TaqSSB mutants are able to form tetramers in solution via arginine-mediated hydrogen-bond interactions that we identified in the crystal packing of wild-type TaqSSB. In EcoSSB, we identified a homologous arginine residue involved in the formation of higher aggregates and metastable highly cooperative single-stranded DNA binding under low salt conditions.     

Identification and properties of the crenarchaeal single-stranded DNA binding protein from Sulfolobus solfataricus

Single-stranded DNA binding proteins (SSBs) play central roles in cellular and viral processes involving the generation of single-stranded DNA. These include DNA replication, homologous recombination and DNA repair pathways. SSBs bind DNA using four ‘OB-fold’ (oligonucleotide/oligosaccharide binding fold) domains that can be organised in a variety of overall quaternary structures. Thus eubacterial SSBs are homotetrameric whilst the eucaryal RPA protein is a heterotrimer and euryarchaeal proteins vary significantly in their subunit compositions. We demonstrate that the crenarchaeal SSB protein is an abundant protein with a unique structural organisation, existing as a monomer in solution and multimerising on DNA binding. The protein binds single-stranded DNA distributively with a binding site size of ~5 nt per monomer. Sulfolobus SSB lacks the zinc finger motif found in the eucaryal and euryarchaeal proteins, possessing instead a flexible C-terminal tail, sensitive to trypsin digestion, that is not required for DNA binding. In comparison with Escherichia coli SSB, the tail may play a role in protein–protein interactions during DNA replication and repair.     

SSB蛋白的高效表达及其与ssDNA的相互作用

大肠杆菌单链结合蛋白SSB在DNA复制、重组和修复中起着重要作用。为研究单链结合蛋白SSB的体外生物功能构建了融合蛋白SSB的表达载体并使其高效表达及易于纯化。ssb基因片段是以E.coli K-12基因组为模板经PCR扩增获得,并通过基因的体外拼接成功构建了表达载体pQE30-ssb。重组菌株M15/ pQE30-ssb经过IPTG的诱导表达了蛋白SSB。收集菌体细胞、超声波破碎后离心取上清进行SDS-PAGE分析,结果表明有一与预期分子量（20.6 kD）相应的诱导表达条带出现,其表达量约占全细胞蛋白的30％且以可溶形式存在。利用固定化金属离子（Ni2+）配体亲和层析柱纯化融合蛋白SSB,其纯度达到90％。通过凝胶层析和等离子共振技术对SSB的生物功能进行了系统研究分析。结果表明,SSB蛋白以四聚体形式与单链DNA分子结合,其亲和力常数（KD）为4.79×10-7 M。     

A role for the human single-stranded DNA binding protein HSSB/RPA in an early stage of nucleotide excision repair.

The human single-stranded DNA binding protein (HSSB/RPA) is involved in several processes that maintain the integrity of the genome including DNA replication, homologous recombination, and nucleotide excision repair of damaged DNA. We report studies that analyze the role of HSSB in DNA repair. Specific protein-protein interactions appear to be involved in the repair function of HSSB, since it cannot be replaced by heterologous single-stranded DNA binding proteins. Anti-HSSB antibodies that inhibit the ability of HSSB to stimulate DNA polymerase alpha also inhibit repair synthesis mediated by human cell-free extracts. However, antibodies that neutralize DNA polymerase alpha do not inhibit repair synthesis. Repair is sensitive to aphidicolin, suggesting that DNA polymerase epsilon or delta participates in nucleotide excision repair by cell extracts. HSSB has a role other than generally stimulating synthesis by DNA polymerases, as it does not enhance the residual damage-dependent background synthesis displayed by repair-deficient extracts from xeroderma pigmentosum cells. Significantly, when damaged DNA is incised by the Escherichia coli UvrABC repair enzyme, human cell extracts can carry out repair synthesis even when HSSB has been neutralized with antibodies. This suggests that HSSB functions in an early stage of repair, rather than exclusively in repair synthesis. A model for the role of HSSB in repair is presented.     

DNA damage detection by an archaeal single-stranded DNA-binding protein

Archaeal DNA repair pathways are not well defined; in particular, there are no convincing candidate proteins for detection of DNA mismatches or the bulky lesions removed by excision repair pathways. Single-stranded DNA-binding proteins (SSBs) play a central role in DNA replication, recombination and repair. The crenarchaeal SSB is a monomer with a single oligonucleotide-binding fold for single-stranded DNA binding coupled to a flexible C-terminal tail reminiscent of bacterial SSB that mediates interactions with other proteins. We demonstrate that Sulfolobus solfataricus SSB can melt DNA containing a mismatch or DNA lesion specifically in vitro. We suggest that a potential role for SSB in archaea is the detection of DNA damage due to local destabilisation of the DNA double helix, followed by recruitment of specific repair proteins. Proteins interacting specifically with a single-stranded DNA:SSB complex include several known or putative DNA repair proteins and DNA helicases.     

The relative rate of synthesis and levels of single-stranded DNA binding protein during induction of SOS repair in Escherichia coli

Summary Induction of the SOS response in Escherichia coli results in an increase in the relative rate of synthesis of single-stranded DNA binding protein (SSB). In contrast to RecA protein, this increase is slow and does not lead to higher SSB levels. The significance of ssb induction to SOS repair is discussed.     

Escherichia coli DinB inhibits replication fork progression without significantly inducing the SOS response

The SOS response is readily triggered by replication fork stalling caused by DNA damage or a dysfunctional replicative apparatus in Escherichia coli cells. E. coli dinB encodes DinB DNA polymerase and its expression is upregulated during the SOS response. DinB catalyzes translesion DNA synthesis in place of a replicative DNA polymerase III that is stalled at a DNA lesion. We showed previously that DNA replication was suppressed without exogenous DNA damage in cells overproducing DinB. In this report, we confirm that this was due to a dose-dependent inhibition of ongoing replication forks by DinB. Interestingly, the DinB-overproducing cells did not significantly induce the SOS response even though DNA replication was perturbed. RecA protein is activated by forming a nucleoprotein filament with single-stranded DNA, which leads to the onset of the SOS response. In the DinB-overproducing cells, RecA was not activated to induce the SOS response. However, the SOS response was observed after heat-inducible activation in strain recA441 (encoding a temperature-sensitive RecA) and after replication blockage in strain dnaE486 (encoding a temperature-sensitive catalytic subunit of the replicative DNA polymerase III) at a non-permissive temperature when DinB was overproduced in these cells. Furthermore, since catalytically inactive DinB could avoid the SOS response to a DinB-promoted fork block, it is unlikely that overproduced DinB takes control of primer extension and thus limits single-stranded DNA. These observations suggest that DinB possesses a feature that suppresses DNA replication but does not abolish the cell's capacity to induce the SOS response. We conclude that DinB impedes replication fork progression in a way that does not activate RecA, in contrast to obstructive DNA lesions and dysfunctional replication machinery.     

Biochemical basis of the constitutive coprotease activity of RecA P67W protein

The mutation of Pro67 to Trp (P67W) in the Escherichia coli RecA protein results in reduced recombination and constitutive coprotease phenotypes. We examined the biochemical properties of this mutant in an effort to understand these altered behaviors. We find that RecA P67W protein can access single-stranded DNA (ssDNA) binding sites within regions of secondary structure more effectively than wild-type protein, and binding to duplex DNA is both faster and more extensive as well. This mutant is also more effective than wild-type RecA protein in displacing SSB protein from ssDNA. An enhancement in SSB protein displacement has been shown previously for RecA441, RecA730, and RecA803 proteins, and similarly, this improved ability to displace SSB protein for RecA P67W protein correlates with an increased rate of association with ssDNA. As for the aforementioned mutant RecA proteins, we expect that this enhanced activity will allow RecA P67W protein to bind ssDNA naturally occurring in undamaged cells and to constitutively induce the SOS response. The DNA strand exchange activity of RecA P67W protein is also altered. Although the rate of duplex DNA uptake into joint molecules is increased compared to that of wild-type RecA protein, the resolution to the nicked circular dsDNA product is reduced. We suggest that either a limited amount of DNA strand reinvasion or a defect in DNA heteroduplex extension is responsible for the impaired recombination ability of this mutant protein.     

RMPs: recombination/replication mediator proteins.

Enzymes for DNA replication and recombination need to gain access to single-stranded DNA (ssDNA) but ssDNA-binding proteins (SSBs) present an obstacle to the formation of enzyme-ssDNA replication and recombination complexes. A specialized class of SSBs, which we designate as recombination/replication mediator proteins (RMPs), promotes enzyme- ssDNA assembly by overcoming SSB inhibition. RMPs exhibit strong conservation of function across divergent species, and display species-specific interactions with SSB and enzymes to neutralize the SSB barrier to enzyme-ssDNA assembly.     

Biochemical characterization of a mutant RecA protein altered in DNA-binding loop 1

The double substitution of Glu156 with Leu and Gly157 with Val in the Escherichia coli RecA protein results in a severely reduced level of recombination and constitutive coprotease behavior. Here we present our examination of the biochemical properties of this mutant protein, RecA N99, in an effort to understand its phenotype and the role of loop 1 (L1) in RecA function. We find that RecA N99 protein has reduced single-stranded DNA (ssDNA)-dependent ATP hydrolysis activity, which is not as sensitive to the presence of SSB protein as wild-type RecA protein. RecA N99 protein is also nearly unable to utilize duplex DNA as a polynucleotide cofactor for ATP hydrolysis, and it shows both a decreased rate of association with ssDNA and a diminished capacity to bind DNA in the secondary binding site. The mutant protein has a corresponding reduction in DNA strand exchange activity, which probably results in the decrease in recombination activity in vivo. The constitutive induction of the SOS response may be a consequence of the impaired ability to repair damaged DNA, resulting in unrepaired ssDNA which can act as a cofactor for the cleavage of LexA repressor. These findings point to an involvement of L1 in both the primary and secondary DNA binding sites of the RecA protein.     

Biochemical characterization of interactions between DNA polymerase and single-stranded DNA-binding protein in bacteriophage RB69

The organization and proper assembly of proteins to the primer-template junction during DNA replication is essential for accurate and processive DNA synthesis. DNA replication in RB69 (a T4-like bacteriophage) is similar to those of eukaryotes and archaea and has been a prototype for studies on DNA replication and assembly of the functional replisome. To examine protein-protein interactions at the DNA replication fork, we have established solution conditions for the formation of a discrete and homogeneous complex of RB69 DNA polymerase (gp43), primer-template DNA, and RB69 single-stranded DNA-binding protein (gp32) using equilibrium fluorescence and light scattering. We have characterized the interaction between DNA polymerase and single-stranded DNA-binding protein and measured a 60-fold increase in the overall affinity of RB69 single-stranded DNA-binding protein (SSB) for template strand DNA in the presence of DNA polymerase that is the result of specific protein-protein interactions. Our data further suggest that the cooperative binding of the RB69 DNA polymerase and SSB to the primer-template junction is a simple but functionally important means of regulatory assembly of replication proteins at the site of action. We have also shown that a functional domain of RB69 single-stranded DNA-binding protein suggested previously to be the site of RB69 DNA polymerase-SSB interactions is dispensable. The data from these studies have been used to model the RB69 DNA polymerase-SSB interaction at the primer-template junction.