Protein modeling and in silico analysis to assess pathogenicity of ABCA4 variants in patients with inherited retinal disease.

Purpose: The retina-specific ABCA transporter, ABCA4, plays an essential role in translocating retinoids required by the visual cycle. ABCA4 genetic variants are known to cause a wide range of inherited retinal disorders, including Stargardt disease and cone-rod dystrophy. More than 1,400 ABCA4 missense variants have been identified; however, more than half of these remain variants of uncertain significance (VUS). The purpose of this study was to employ a predictive strategy to assess the pathogenicity of ABCA4 variants in inherited retinal diseases using protein modeling and computational approaches. Methods: We studied 13 clinically well-defined patients with ABCA4 retinopathies and identified the presence of 10 missense variants, including one novel variant in the ABCA4 gene, by next-generation sequencing (NGS). All variants were structurally analyzed using AlphaFold2 models and existing experimental structures of human ABCA4 protein. The results of these analyses were compared with patient clinical presentations to test the effectiveness of the methods employed in predicting variant pathogenicity. Results: We conducted a phenotype-genotype comparison of 13 genetically and phenotypically well-defined retinal disease patients. The in silico protein structure analyses we employed successfully detected the deleterious effect of missense variants found in this affected patient cohort. Our study provides American College of Medical Genetics and Genomics (ACMG)-defined supporting evidence of the pathogenicity of nine missense ABCA4 variants, aligning with the observed clinical phenotypes in this cohort. Conclusions: In this report, we describe a systematic approach to predicting the pathogenicity of ABCA4 variants by means of three-dimensional (3D) protein modeling and in silico structure analysis. Our results demonstrate concordance between disease severity and structural changes in protein models induced by genetic variations. Furthermore, the present study suggests that in silico protein structure analysis can be used as a predictor of pathogenicity and may facilitate the assessment of genetic VUS.

Human papillomavirus spectrum of HPV-infected women in Nigeria: an analysis by next-generation sequencing and type-specific PCR.

BACKGROUND: Human papillomavirus (HPV) infection and cervical cancer are leading health problems and causes of death in many parts of the world. There are ~ 200 HPV types that can infect humans. This study aims to understand the spectrum of HPV infections in Nigerian women with normal or abnormal cytology. METHODS: We screened cervical samples from 90 women with possible HPV infections collected in two regional hospitals in Nigeria. The first screening was done using next-generation DNA sequencing (NGS), identifying multiple HPV types in many samples. Thereafter, type-specific PCR analysis was used to verify the NGS-identified HPV types in each sample. RESULTS: NGS analysis of the 90 samples from the Nigerian cohort identified 44 HPV types. The type-specific PCR confirmed 25 HPV types out of the 44 HPV types detected by NGS, and ~ 10 of these types were the most prevalent. The top five prevalent types found in the Nigerian cohort were HPV71 (17%), HPV82 (15%), HPV16 (16%), HPV6 (10%), and HPV20 (7%). Among the PCR-confirmed HPV types, we found 40.98% high-risk HPV types, 27.22% low-risk HPV types, and 31.15% undetermined HPV types. Among these 25 HPV types in Nigeria, only six were included in the current nine-valent HPV vaccine. We also observed strikingly high multiple HPV infections in most patients, with as many as nine HPV types in a few single samples. CONCLUSIONS: Our NGS-PCR approach of HPV typing in the Nigerian cohort samples unveiled all possible HPV types currently circulating in Nigerian people. We confirmed 25 HPV types using NGS and PCR, with many samples infected with multiple HPV types. However, only six of these types are part of the nine-valent HPV vaccines indicating the need to develop region-specific selective vaccines.

Protein-DNA Interactions Regulate Human Papillomavirus DNA Replication, Transcription, and Oncogenesis.

Human papillomavirus (HPV) is a group of alpha papillomaviruses that cause various illnesses, including cancer. There are more than 160 types of HPV, with many being "high-risk" types that have been clinically linked to cervical and other types of cancer. "Low-risk" types of HPV cause less severe conditions, such as genital warts. Over the past few decades, numerous studies have shed light on how HPV induces carcinogenesis. The HPV genome is a circular double-stranded DNA molecule that is approximately 8 kilobases in size. Replication of this genome is strictly regulated and requires two virus-encoded proteins, E1 and E2. E1 is a DNA helicase that is necessary for replisome assembly and replication of the HPV genome. On the other hand, E2 is responsible for initiating DNA replication and regulating the transcription of HPV-encoded genes, most importantly the E6 and E7 oncogenes. This article explores the genetic characteristics of high-risk HPV types, the roles of HPV-encoded proteins in HPV DNA replication, the regulation of transcription of E6 and E7 oncogenes, and the development of oncogenesis.

Sequence-Dependent Interaction of the Human Papillomavirus E2 Protein with the DNA Elements on Its DNA Replication Origin.

The human papillomavirus (HPV) E2 protein is essential for regulating the initiation of viral DNA replication as well as the regulation of transcription of certain HPV-encoded genes. Its ability to recognize and bind to its four recognition sequences in the viral origin is a key step in the initiation of HPV DNA replication. Thus, understanding the mechanism of DNA binding by E2 protein and the unique roles played by individual DNA sequence elements of the replication origin is essential. We have purified the recombinant full-length HPV type 11 E2 protein. Quantitative DNA binding analysis indicated E2 protein bound all four DNA binding sites with reasonably high affinities but with distinct preferences. It bound its cognate binding sites 1, 2, and 4 with higher affinities, but bound binding site 3 with lower affinity. Analysis of binding to these sites unraveled multiple sequence elements that appeared to influence E2 binding affinity and target discrimination, including the sequence of spacer region, flanking sequences, and proximity of E2 binding sites. Thermodynamic analysis indicated hydrophobic interaction in the protein-DNA complex formation. Our studies indicate a large multi-protein complex formation on the HPV-origin DNA, likely due to reasonably high binding affinities as well as intrinsic oligomerization propensity of E2 dimers.

Structural and Pathogenic Impacts of ABCA4 Variants in Retinal Degenerations-An In-Silico Study.

The retina-specific ATP-binding cassette transporter protein ABCA4 is responsible for properly continuing the visual cycle by removing toxic retinoid byproducts of phototransduction. Functional impairment caused by ABCA4 sequence variations is the leading cause of autosomal recessive inherited retinal disorders, including Stargardt disease, retinitis pigmentosa, and cone-rod dystrophy. To date, more than 3000 ABCA4 genetic variants have been identified, approximately 40 percent of which have not been able to be classified for pathogenicity assessments. This study examined 30 missense ABCA4 variants using AlphaFold2 protein modeling and computational structure analysis for pathogenicity prediction. All variants classified as pathogenic (n = 10) were found to have deleterious structural consequences. Eight of the ten benign variants were structurally neutral, while the remaining two resulted in mild structural changes. This study's results provided multiple lines of computational pathogenicity evidence for eight ABCA4 variants of uncertain clinical significance. Overall, in silico analyses of ABCA4 can provide a valuable tool for understanding the molecular mechanisms of retinal degeneration and their pathogenic impact.

Functional significance of the conserved C-Terminal VFVNFA motif in the retina-specific ABC transporter, ABCA4, and its role in inherited visual disease.

The human retina-specific ATP binding cassette transporter, ABCA4, plays a significant role in the visual cycle. Mutations in the ABCA4 gene result in a broad spectrum of severe, blinding, retinal degenerative diseases, including Stargardt macular dystrophy, fundus flavimaculatus, autosomal recessive (ar)-retinitis pigmentosa, and ar-cone-rod dystrophy. Genetic testing frequently yields novel variants of unknown significance, making accurate prognosis and therapeutic approaches difficult. Recently, we have reported a novel variant of ABCA4 corresponding to a four-nucleotide deletion which led to a premature stop codon and loss of the last 161 amino acids, including the highly-conserved VFVNFA motif. Despite the presence of this motif among other ABCA proteins, knowledge of the functional significance of this sequence remains limited. In this study, we have conducted structural and functional analyses of recombinant ABCA4 polypeptides with altered VFVNFA motifs to evaluate the importance of this sequence. Further investigation of ABCA4 subdomain interactions, using Fluorescence Resonance Energy Transfer, demonstrated a loss of interaction between nucleotide binding domains in the absence of the VFVNFA motif.

Genetic variations in the DNA replication origins of human papillomavirus family correlate with their oncogenic potential.

Human papillomaviruses (HPVs) encompass a large family of viruses that range from benign to highly carcinogenic. The crucial differences between benign and carcinogenic types of HPV remain unknown, except that the two HPV types differ in the frequency of DNA replication. We have systematically analyzed the mechanism of HPV DNA replication initiation in low-risk and high-risk HPVs. Our results demonstrate that HPV-encoded E2 initiator protein and its four binding sites in the replication origin play pivotal roles in determining the destiny of the HPV-infected cell. We have identified strain-specific single nucleotide variations in E2 binding sites found only in the high-risk HPVs. We have demonstrated that these variations result in attenuated formation of the E2-DNA complex. E2 binding to these sites is linked to the activation of the DNA replication origin as well as initiation of DNA replication. Both electrophoretic mobility shift assay and atomic force microscopy studies demonstrated that binding of E2 from either low- or high-risk HPVs with variant binding sequences lacked multimeric E2-DNA complex formation in vitro. These results provided a molecular basis of differential DNA replication in the two types of HPVs and pointed to a correlation with the development of cancer.

Multiple conformational states of DnaA protein regulate its interaction with DnaA boxes in the initiation of DNA replication.

DnaA protein is the initiator of genomic DNA replication in prokaryotes. It binds to specific DNA sequences in the origin of DNA replication and unwinds small AT-rich sequences downstream for the assembly of the replisome. The mechanism of activation of DnaA that enables it to bind and organize the origin DNA and leads to replication initiation remains unclear. In this study, we have developed double-labeled fluorescent DnaA probes to analyze conformational states of DnaA protein upon binding DNA, nucleotide, and Soj sporulation protein using Fluorescence Resonance Energy Transfer (FRET). Our studies demonstrate that DnaA protein undergoes large conformational changes upon binding to substrates and there are multiple distinct conformational states that enable it to initiate DNA replication. DnaA protein adopted a relaxed conformation by expanding ~15Å upon binding ATP and DNA to form the ATP·DnaA·DNA complex. Hydrolysis of bound ATP to ADP led to a contraction of DnaA within the complex. The relaxed conformation of DnaA is likely required for the formation of the multi-protein ATP·DnaA·DNA complex. In the initiation of sporulation, Soj binding to DnaA prevented relaxation of its conformation. Soj·ADP appeared to block the activation of DnaA, suggesting a mechanism for Soj·ADP in switching initiation of DNA replication to sporulation. Our studies demonstrate that multiple conformational states of DnaA protein regulate its binding to DNA in the initiation of DNA replication.

Retinoid binding properties of nucleotide binding domain 1 of the Stargardt disease-associated ATP binding cassette (ABC) transporter, ABCA4.

The retina-specific ATP binding cassette transporter, ABCA4 protein, is associated with a broad range of inherited macular degenerations, including Stargardt disease, autosomal recessive cone rod dystrophy, and fundus flavimaculatus. In order to understand its role in retinal transport in rod out segment discs, we have investigated the interactions of the soluble domains of ABCA4 with both 11-cis- and all-trans-retinal. Using fluorescence anisotropy-based binding analysis and recombinant polypeptides derived from the amino acid sequences of the four soluble domains of ABCA4, we demonstrated that the nucleotide binding domain 1 (NBD1) specifically bound 11-cis-retinal. Its affinity for all-trans-retinal was markedly reduced. Stargardt disease-associated mutations in this domain resulted in attenuation of 11-cis-retinal binding. Significant differences in 11-cis-retinal binding affinities were observed between NBD1 and other cytoplasmic and lumenal domains of ABCA4. The results suggest a possible role of ABCA4 and, in particular, the NBD1 domain in 11-cis-retinal binding. These results also correlate well with a recent report on the in vivo role of ABCA4 in 11-cis-retinal transport.

Thermodynamic analysis of DNA binding by a Bacillus single stranded DNA binding protein.

BACKGROUND: Single-stranded DNA binding proteins (SSB) are essential for DNA replication, repair, and recombination in all organisms. SSB works in concert with a variety of DNA metabolizing enzymes such as DNA polymerase. RESULTS: We have cloned and purified SSB from Bacillus anthracis (SSB(BA)). In the absence of DNA, at concentrations ≤100 µg/ml, SSB(BA) did not form a stable tetramer and appeared to resemble bacteriophage T4 gene 32 protein. Fluorescence anisotropy studies demonstrated that SSB(BA) bound ssDNA with high affinity comparable to other prokaryotic SSBs. Thermodynamic analysis indicated both hydrophobic and ionic contributions to ssDNA binding. FRET analysis of oligo(dT)(70) binding suggested that SSB(BA) forms a tetrameric assembly upon ssDNA binding. This report provides evidence of a bacterial SSB that utilizes a novel mechanism for DNA binding through the formation of a transient tetrameric structure. CONCLUSIONS: Unlike other prokaryotic SSB proteins, SSB(BA) from Bacillus anthracis appeared to be monomeric at concentrations ≤100 µg/ml as determined by SE-HPLC. SSB(BA) retained its ability to bind ssDNA with very high affinity, comparable to SSB proteins which are tetrameric. In the presence of a long ssDNA template, SSB(BA) appears to form a transient tetrameric structure. Its unique structure appears to be due to the cumulative effect of multiple key amino acid changes in its sequence during evolution, leading to perturbation of stable dimer and tetramer formation. The structural features of SSB(BA) could promote facile assembly and disassembly of the protein-DNA complex required in processes such as DNA replication.

Interaction of extracellular domain 2 of the human retina-specific ATP-binding cassette transporter (ABCA4) with all-trans-retinal.

The retina-specific ATP-binding cassette (ABC) transporter, ABCA4, is essential for transport of all-trans-retinal from the rod outer segment discs in the retina and is associated with a broad range of inherited retinal diseases, including Stargardt disease, autosomal recessive cone rod dystrophy, and fundus flavimaculatus. A unique feature of the ABCA subfamily of ABC transporters is the presence of highly conserved, long extracellular loops or domains (ECDs) with unknown function. The high degree of sequence conservation and mapped disease-associated mutations in these domains suggests an important physiological significance. Conformational analysis using CD spectroscopy of purified, recombinant ECD2 protein demonstrated that it has an ordered and stable structure composed of 27 +/- 3% alpha-helix, 20 +/- 3% beta-pleated sheet, and 53 +/- 3% coil. Significant conformational changes were observed in disease-associated mutant proteins. Using intrinsic tryptophan fluorescence emission spectrum of ECD2 polypeptide and fluorescence anisotropy, we have demonstrated that this domain specifically interacts with all-trans-retinal. Furthermore, the retinal interaction appeared preferential for the all-trans-isomer and was directly measurable through fluorescence anisotropy analysis. Our results demonstrate that the three macular degeneration-associated mutations lead to significant changes in the secondary structure of the ECD2 domain of ABCA4, as well as in its interaction with all-trans-retinal.

An essential DnaB helicase of Bacillus anthracis: identification, characterization, and mechanism of action.

We have described a novel essential replicative DNA helicase from Bacillus anthracis, the identification of its gene, and the elucidation of its enzymatic characteristics. Anthrax DnaB helicase (DnaB(BA)) is a 453-amino-acid, 50-kDa polypeptide with ATPase and DNA helicase activities. DnaB(BA) displayed distinct enzymatic and kinetic properties. DnaB(BA) has low single-stranded DNA (ssDNA)-dependent ATPase activity but possesses a strong 5'-->3' DNA helicase activity. The stimulation of ATPase activity appeared to be a function of the length of the ssDNA template rather than of ssDNA binding alone. The highest specific activity was observed with M13mp19 ssDNA. The results presented here indicated that the ATPase activity of DnaB(BA) was coupled to its migration on an ssDNA template rather than to DNA binding alone. It did not require nucleotide to bind ssDNA. DnaB(BA) demonstrated a strong DNA helicase activity that required ATP or dATP. Therefore, DnaB(BA) has an attenuated ATPase activity and a highly active DNA helicase activity. Based on the ratio of DNA helicase and ATPase activities, DnaB(BA) is highly efficient in DNA unwinding and its coupling to ATP consumption.

Mechanisms of DNA binding and regulation of Bacillus anthracis DNA primase.

DNA primases are pivotal enzymes in chromosomal DNA replication in all organisms. In this article, we report unique mechanistic characteristics of recombinant DNA primase from Bacillus anthracis. The mechanism of action of B. anthracis DNA primase (DnaG(BA)) may be described in several distinct steps as follows. Its mechanism of action is initiated when it binds to single-stranded DNA (ssDNA) in the form of a trimer. Although DnaG(BA) binds to different DNA sequences with moderate affinity (as expected of a mobile DNA binding protein), we found that DnaG(BA) bound to the origin of bacteriophage G4 (G4ori) with approximately 8-fold higher affinity. DnaG(BA) was strongly stimulated (>or=75-fold) by its cognate helicase, DnaB(BA), during RNA primer synthesis. With the G4ori ssDNA template, DnaG(BA) formed short (

Discovery, characterization and comparison of inhibitors of Bacillus anthracis and Staphylococcus aureus replicative DNA helicases.

Antibacterial compounds with new mechanisms of action are needed for effective therapy against drug-resistant pathogens in the clinic and in biodefense. Screens for inhibitors of the essential replicative helicases of Bacillus anthracis and Staphylococcus aureus yielded 18 confirmed hits (IC(50)25 microM). Several (5 of 18) of the inhibitors were also shown to inhibit DNA replication in permeabilized polA-deficient B. anthracis cells. One of the most potent inhibitors also displayed antibacterial activity (MIC approximately 5 microg/ml against a range of Gram-positive species including bacilli and staphylococci) together with good selectivity for bacterial versus mammalian cells (CC(50)/MIC>16) suitable for further optimization. This compound shares the bicyclic ring of the clinically proven aminocoumarin scaffold, but is not a gyrase inhibitor. It exhibits a mixed mode of helicase inhibition including a component of competitive inhibition with the DNA substrate (K(i)=8 microM) and is rapidly bactericidal at 4 x MIC.

Site-specific fluorescence double-labeling of proteins and analysis of structural changes in solution by Fluorescence Resonance Energy Transfer (FRET).

Fluorescence Resonance Energy Transfer (FRET) is a well-known methodology for detection and quantitation of structural changes of proteins in solution. FRET requires site-specific protein labeling with two fluorophores, one of which functions as an energy donor and the other one as an energy acceptor. However, the site-specific labeling of protein is often complex and difficult, particularly when inserting two fluorophores in specific sites. We have examined several protein labeling approaches with a varying degree of success. Described here is a dual labeling strategy that worked reproducibly in a number of protein targets and we believe will be applicable to a variety of proteins, which have few or no native cysteine (Cys) residues. We have successfully double-labeled DnaA protein of Bacillus anthracis, which lacks intrinsic Cys residues. A cysteine residue was inserted at the N-terminus by in vitro mutagenesis and a Cys-Cys-Phe-Gly-Cys-Cys (CCPGCC) sequence at the C-terminus by PCR. This protein was labeled site-specifically with a fluorescein derivative, FlAsH, at the CCPGCC sequence followed by Alexa568 maleimide at the N-terminus Cys residue. Structural changes of the protein with nucleotide, DNA and an inhibitor protein binding were determined by FRET analysis of the double-labeled protein. This comprehensive novel methodology for site-specific protein labeling with different fluorophores is applicable for understanding different in vitro proteomic structural studies. Here, we describe a verified technique used for FRET spectral analysis and quantitative evaluation of structural changes using fluorophore labeled DnaA protein constructs as an example.

Modulation of enzymatic activities of Escherichia coli DnaB helicase by single-stranded DNA-binding proteins.

The modulation of enzymatic activities of Escherichia coli DnaB helicase by homologous and heterologous single-stranded DNA-binding proteins (SSBs) and its DNA substrates were analyzed. Although DnaB helicase can unwind a variety of DNA substrates possessing different fork-like structures, the rate of DNA unwinding was significantly diminished with substrates lacking a 3' fork. A 5 nt fork appeared to be adequate to attain the maximum rate of DNA unwinding. Efficient helicase action of DnaB requires the participation of SSBs. Studies involving heterologous SSBs demonstrated that they can stimulate the helicase activity of DnaB protein under certain conditions. However, this stimulation occurs in a manner distinctly different from that observed with cognate E.coli SSB. The E.coli SSB was found to stimulate the helicase activity over a wide range of SSB concentrations and was unique in its strong inhibition of single-stranded DNA-dependent ATPase activity when uncoupled from the DNA helicase activity. In the presence of a helicase substrate, the ATPase activity of DnaB helicase remained uninhibited. Thus, E.coli SSB appears to coordinate and couple the ATPase activity to the DNA helicase activity by suppressing unproductive ATP hydrolysis by DnaB helicase.

Subunit interactions in the assembly of Saccharomyces cerevisiae DNA polymerase alpha.

Eukaryotic DNA polymerase (pol) alpha is a complex of four subunits. The subunits in the yeast Saccharomyces cerevisiae are: 167, 79, 62 and 48 kDa polypeptides. The p79 subunit has no known enzymatic functions, but it is essential for growth and chromosomal DNA replication. We have analyzed the interaction between the subunits of yeast pol alpha, particularly p167 and p79, using a yeast two-hybrid screen and deletion analysis. We have identified the interaction sites in each of these two subunits leading to p167.p79 complex formation, and correlated our results with the available genetic data. A detailed two-hybrid analysis, using the p79 gene as the bait and a yeast genomic DNA library, identified two independent groups of positive clones. One group that displayed strong positive interaction (delta1) with p79 represented a fusion of the p167 open reading frame at 3502 bp (Ile1168), and the second group, displaying weak positive interaction (delta2) with p79, had a fusion at 3697 bp (Asn1233) with the DNA-binding domain of the yeast Gal4 transcription factor. A detailed deletion analysis of the downstream region indicated the existence of two subdomains that interact with p79. Subdomain I encompasses a 65 amino acid segment between Ile1168 and Phe1232, and subdomain II is a 25 amino acid segment between Glu1259 and Leu1283. Deletion and two-hybrid interaction analysis of the p79 subunit of pol alpha revealed a complementary region with two subdomains: a 67 amino acid segment between Asn189 and Gln255 (I) and a 68 amino acid segment between Glu256 and Met323 (II). The p79 subdomains I and II appeared to interact with the p167 subdomains I and II, respectively. Analysis of interaction between p62 and various deletion clones of p167 did not result in an unambiguous and stable positive interaction in the two-hybrid screen between these two subunits. A strong interaction between p167 and p62 would probably require the presence of either p79 or p48 in the complex.

Quantitative analysis of nucleotide modulation of DNA binding by DnaC protein of Escherichia coli.

In this study, we have presented the first report of Escherichia coli DnaC protein binding to ssDNA (single stranded DNA) in an apparent hexameric form. DnaC protein transfers DnaB helicase onto a nascent chromosomal DNA replication fork at oriC, the origin of E. coli DNA replication. In eukaryotes, Cdc6 protein may play a similar role in the DNA helicase loading in the replication fork during replication initiation at the origin. We have analysed the DNA-binding properties of DnaC protein and a quantitative analysis of the nucleotide regulation of DnaC-DNA and DnaC-DnaB interactions using fluorescence anisotropy and affinity sensor analysis. DnaC protein bound to ssDNA with low to moderate affinity and the affinity was strictly modulated by nucleotides. DnaC bound ssDNA in the complete absence of nucleotides. The DNA-binding affinity was significantly increased in the presence of ATP, but not ATP[S]. In the presence of ADP, the binding affinity decreased approximately fifty-fold. Both anisotropy and biosensor analyses demonstrated that with DnaC protein, ATP facilitated ssDNA binding, whereas ADP facilitated its dissociation from ssDNA, which is a characteristic of an ATP/ADP switch. Both ssDNA and nucleotides modulate DnaB6*DnaC6 complex formation, which has significant implications in DnaC protein function. Based on the thermodynamic data provided in this study, we have proposed a mechanism of DnaB loading on to ssDNA by DnaC protein.

Quantitative analysis of the mechanism of DNA binding by Bacillus DnaA protein.

DnaA protein has the sole responsibility of initiating a new round of DNA replication in prokaryotic organisms. It recognizes the origin of DNA replication, and initiates chromosomal DNA replication in the bacterial genome. In Gram-negative Escherichia coli, a large number of DnaA molecules bind to specific DNA sequences (known as DnaA boxes) in the origin of DNA replication, oriC, leading to the activation of the origin. We have cloned, expressed, and purified full-length DnaA protein in large quantity from Gram-positive pathogen Bacillus anthracis (DnaA(BA)). DnaA(BA) was a highly soluble monomeric protein making it amenable to quantitative analysis of its origin recognition mechanisms. DnaA(BA) bound DnaA boxes with widely divergent affinities in sequence and ATP-dependent manner. In the presence of ATP, the K(D) ranged from 3.8 × 10(-8) M for a specific DnaA box sequence to 4.1 × 10(-7) M for a non-specific DNA sequence and decreased significantly in the presence of ADP. Thermodynamic analyses of temperature and salt dependence of DNA binding indicated that hydrophobic (entropic) and ionic bonds contributed to the DnaA(BA)·DNA complex formation. DnaA(BA) had a DNA-dependent ATPase activity. DNA sequences acted as positive effectors and modulated the rate (V(max)) of ATP hydrolysis without any significant change in ATP binding affinity.

Quantitative analysis of binding of single-stranded DNA by Escherichia coli DnaB helicase and the DnaB x DnaC complex.

DnaB helicase is responsible for unwinding duplex DNA during chromosomal DNA replication and is an essential component of the DNA replication apparatus in Escherichia coli. We have analyzed the mechanism of binding of single-stranded DNA (ssDNA) by the DnaB x DnaC complex and DnaB helicase. Binding of ssDNA to DnaB helicase was significantly modulated by nucleotide cofactors, and the modulation was distinctly different for its complex with DnaC. DnaB helicase bound ssDNA with a high affinity [Kd = (5.09 +/- 0.32) x 10(-8) M] only in the presence of ATPgammaS, a nonhydrolyzable analogue of ATP, but not other nucleotides. The binding was sensitive to ionic strength but not to changes in temperature in the range of 30-37 degrees C. On the other hand, ssDNA binding in the presence of ADP was weaker than that observed with ATPgammaS, and the binding was insensitive to ionic strength. DnaC protein hexamerizes to form a 1:1 complex with the DnaB hexamer and loads it onto the ssDNA by forming a DnaB6 x DnaC6 dodecameric complex. Our results demonstrate that the DnaB6 x DnaC6 complex bound ssDNA with a high affinity [Kd = (6.26 +/- 0.65) x 10(-8) M] in the presence of ATP, unlike the DnaB hexamer. In the presence of ATPgammaS or ADP, binding of ssDNA by the DnaB6 x DnaC6 complex was a lower-affinity process. In summary, our results suggest that in the presence of ATP in vivo, the DnaB6 x DnaC6 complex should be more efficient in binding DNA as well as in loading DnaB onto the ssDNA than DnaB helicase itself.