Nucleotide binding domain 1 of the human retinal ABC transporter functions as a general ribonucleotidase.

Members of the ATP binding cassette (ABC) superfamily are transmembrane proteins that are found in a variety of tissues which transport substances across cell membranes in an energy-dependent manner. The retina-specific ABC protein (ABCR) has been linked through genetic studies to a number of inherited visual disorders, including Stargardt macular degeneration and age-related macular degeneration (ARMD). Like other ABC transporters, ABCR is characterized by two nucleotide binding domains and two transmembrane domains. We have cloned and expressed the 522-amino acid (aa) N-terminal cytoplasmic region (aa 854-1375) of ABCR containing nucleotide binding domain 1 (NBD1) with a purification tag at its amino terminus. The expressed recombinant protein was found to be soluble and was purified using single-step affinity chromatography. The purified protein migrated as a 66 kDa protein on SDS-PAGE. Analysis of the ATP binding and hydrolysis properties of the NBD1 polypeptide demonstrated significant differences between NBD1 and NBD2 [Biswas, E. E., and Biswas, S. B. (2000) Biochemistry 39, 15879-15886]. NBD1 was active as an ATPase, and nucleotide inhibition studies suggested that nucleotide binding was not specific for ATP and all four ribonucleotides can compete for binding. Further analysis demonstrated that NBD1 is a general nucleotidase capable of hydrolysis of ATP, CTP, GTP, and UTP. In contrast, NBD2 is specific for adenosine nucleotides (ATP and dATP). NBD1 bound ATP with a higher affinity than NBD2 (K(mNBD1) = 200 microm vs K(mNBD2) = 631 microm) but was less efficient as an ATPase (V(maxNBD1) = 28.9 nmol min(-)(1) mg(-)(1) vs V(maxNBD2) = 144 nmol min(-)(1) mg(-)(1)). The binding efficiencies for CTP and GTP were comparable to that observed for ATP (K(mCTP) = 155 microm vs K(mGTP) = 183 microm), while that observed for UTP was decreased 2-fold (K(mUTP) = 436 microm). Thus, the nucleotide binding preference of NBD1 is as follows: CTP > GTP > ATP >> UTP. These studies demonstrate that NBD1 of ABCR is a general nucleotidase, whereas NBD2 is a specific ATPase.

A novel human hexameric DNA helicase: expression, purification and characterization.

We have cloned, expressed and purified a hexameric human DNA helicase (hHcsA) from HeLa cells. Sequence analysis demonstrated that the hHcsA has strong sequence homology with DNA helicase genes from Saccharomyces cerevisiae and Caenorhabditis elegans, indicating that this gene appears to be well conserved from yeast to human. The hHcsA gene was cloned and expressed in Escherichia coli and purified to homogeneity. The expressed protein had a subunit molecular mass of 116 kDa and analysis of its native molecular mass by size exclusion chromatography suggested that hHcsA is a hexameric protein. The hHcsA protein had a strong DNA-dependent ATPase activity that was stimulated >/=5-fold by single-stranded DNA (ssDNA). Human hHcsA unwinds duplex DNA and analysis of the polarity of translocation demonstrated that the polarity of DNA unwinding was in a 5'-->3' direction. The helicase activity was stimulated by human and yeast replication protein A, but not significantly by E.coli ssDNA-binding protein. We have analyzed expression levels of the hHcsA gene in HeLa cells during various phases of the cell cycle using in situ hybridization analysis. Our results indicated that the expression of the hHcsA gene, as evidenced from the mRNA levels, is cell cycle-dependent. The maximal level of hHcsA expression was observed in late G(1)/early S phase, suggesting a possible role for this protein during S phase and in DNA synthesis.

The C-terminal nucleotide binding domain of the human retinal ABCR protein is an adenosine triphosphatase.

The rod outer segment ATP binding cassette (ABC) transporter protein (ABCR) plays an important role in retinal rod cells presumably transporting retinal. Genetic studies in humans have linked mutations in the ABCR gene to a number of inherited retinal diseases particularly Stargardt macular degeneration and age-related macular degeneration (ARMD). The ABCR protein is characterized by two nucleotide binding domains and two transmembrane domains, each consisting of six membrane-spanning helices. We have cloned and expressed the 376 amino acid (aa) C-terminal end of this protein (amino acid residues 1898-2273) containing the second nucleotide binding domain (NBD2) with a purification tag at its amino terminus. The expressed protein was found to be soluble and was purified using a rapid and high-yield single-step procedure. The purified protein was monomeric and migrated as a 43 kDa protein in SDS-PAGE. The purified NBD2 protein had strong ATPase activity with a K(m) of 631 microM and V(max) of 144 nmol min(-1) mg(-1). This ATPase activity on normalization was kinetically comparable to that observed for purified and reconstituted native ABCR. Nucleotide inhibition studies suggest that the binding of NBD2 is specific for ATP/dATP, and that none of the other ribonucleotides appeared to compete for binding at this site. These studies demonstrate that cloned and expressed NBD2 protein is a fully functional ATPase in the absence of the remainder of the molecule. The level of ATPase activity was comparable to that of trans-retinal-stimulated ABCR ATPase. The NBD2 expression plasmid was used to generate a Leu2027Phe mutation associated with Stargardt disease. Analysis of the ATPase activity of the mutant protein demonstrated that it had a 14-fold increase in binding affinity (K(m) = 46 microM) with a corresponding 9-fold decrease in the rate of hydrolysis (V(max) = 16.6 nmol min(-1) mg(-1)), indicating a significant alteration of the ATPase function. It also provided a molecular basis of Stargardt disease involving this mutation.

Mechanism of DnaB helicase of Escherichia coli: structural domains involved in ATP hydrolysis, DNA binding, and oligomerization.

We describe the delineation of three distinct structural domains of the DnaB helicase of Escherichia coli: domain alpha, amino acid residues (aa) 1-156; domain beta, aa 157-302; and domain gamma, aa 303-471. Using mutants with deletion in these domains, we have examined their role(s) in hexamer formation, DNA-dependent ATPase, and DNA helicase activities. The mutant DnaBbetagamma protein, in which domain alpha was deleted, formed a hexamer; whereas the mutant DnaBalphabeta, in which domain gamma was deleted, could form only dimers. The dimerization of DnaBalphabeta was Mg(2+) dependent. These data suggest that the oligomerization of DnaB helicase involves at least two distinct protein-protein interaction sites; one of these sites is located primarily within domain beta (site 1), while the other interaction site is located within domain gamma (site 2). The mutant DnaBbeta, a polypeptide of 147 aa, where both domains alpha and gamma were deleted, displayed a completely functional ATPase activity. This domain, thus, constitutes the "central catalytic domain" for ATPase activity. The ATPase activity of DnaBalphabeta was kinetically comparable to that of DnaBbeta, indicating that domain alpha had little or no influence on the ATPase activity. In both cases, the ATPase activities were DNA independent. DnaBbetagamma had a DNA-dependent ATPase activity that was kinetically comparable to the ATPase activity of wild-type DnaB protein (wtDnaB), indicating a specific role for C-terminal domain gamma in enhancement of the ATPase activity of domain beta as well as in DNA binding. Mutant DnaBbetagamma, which lacked domain alpha, was devoid of any helicase activity pointing to a significant role for domain alpha. The major findings of this study are (i) domain beta contained a functional ATPase active site; (ii) domain gamma appeared to be the DNA binding domain and a positive regulator of the ATPase activity of domain beta; (iii) although domain alpha did not have any significant effect on the ATPase, DNA binding activities, or hexamer formation, it definitely plays a pivotal role in transducing the energy of ATP hydrolysis to DNA unwinding by the hexamer; and (iv) all three domains are required for helicase activity.

Mechanism of DNA binding by the DnaB helicase of Escherichia coli: analysis of the roles of domain gamma in DNA binding.

We have analyzed the mechanism of single-stranded DNA (ssDNA) binding mediated by the C-terminal domain gamma of the DnaB helicase of Escherichia coli. Sequence analysis of this domain indicated a specific basic region, "RSRARR", and a leucine zipper motif that are likely involved in ssDNA binding. We have carried out deletion as well as in vitro mutagenesis of specific amino acid residues in this region in order to determine their function(s) in DNA binding. The functions of the RSRARR domain in DNA binding were analyzed by site-directed mutagenesis. DnaBMut1, with mutations R(328)A and R(329)A, had a significant decrease in the DNA dependence of ATPase activity and lost its DNA helicase activity completely, indicating the important roles of these residues in DNA binding and helicase activities. DnaBMut2, with mutations R(324)A and R(326)A, had significantly attenuated DNA binding as well as DNA-dependent ATPase and DNA helicase activities, indicating that these residues also play a role in DNA binding and helicase activities. The role(s) of the leucine zipper dimerization motif was (were) determined by deletion analysis. The DnaB Delta 1 mutant with a 55 amino acid C-terminal deletion, which left the leucine zipper and basic DNA binding regions intact, retained DNA binding as well as DNA helicase activities. However, the DnaB Delta 2 mutant with a 113 amino acid C-terminal deletion that included the leucine zipper dimerization motif, but not the RSRARR sequence, lost DNA binding, DNA helicase activities, and hexamer formation. The major findings of this study are (i) the leucine zipper dimerization domain, I(361)-L(389), is absolutely required for (a) dimerization and (b) ssDNA binding; (ii) the base-rich RSRARR sequence is required for DNA binding; (iii) three regions of domain gamma (gamma I, gamma II, and gamma III) differentially regulate the ATPase activity; (iv) there are likely three ssDNA binding sites per hexamer; and (v) a working model of DNA unwinding by the DnaB hexamer is proposed.

Purification and characterization of DNA polymerase alpha-associated replication protein A-dependent yeast DNA helicase A.

A novel, eukaryotic, hexameric DNA helicase that was earlier identified as a component of the multiprotein polymerase alpha complex [Biswas et al. (1993) Biochemistry 32, 13393-13398] has been purified to homogeneity and characterized. Thus far, our studies demonstrated that helicase A shares certain unique features of two other hexameric DNA helicases: the DnaB helicase of Escherichia coli and the T-antigen helicase of the SV40 virus. The helicase activity was stimulated by yeast replication protein A (RPA) and to a lower extent by E. coli single-stranded DNA binding protein (SSB). The helicase had an apparent molecular mass of 90 kDa, as determined by its mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A tryptic peptide fragment of the polypeptide was sequenced followed by a BLAST search of GenBank with the tryptic peptide sequence. The search identified a 1.8 kb open reading frame previously designated as ykl017c on chromosome XI, that codes for a 78.3 kDa (683 amino acid) polypeptide. The important features of the polypeptide sequence of helicase A included a type I ATP/GTP binding motif, and a K E E R R L N V A M T R P R R sequence at the C-terminus that may be indicative of a nuclear localization signal which is required of a nuclear DNA helicase. The polypeptide sequence of helicase A appears to have homology to the DnaB helicase of E. coli (approximately 25%). The facts that these two helicases are vastly separated by evolution and retained similar structural and functional features, as demonstrated here, point to a possible significance of this limited homology. Although the amount of purified helicase A was limited, we have carried out necessary enzymatic characterization so that these data could be correlated with that of immunoaffinity-purified helicase A and recombinant helicase A expressed in heterologous systems.

Yeast DNA helicase A: cloning, expression, purification, and enzymatic characterization.

We have cloned and expressed the yeast DNA helicase A in Escherichia coli at a high level (approximately 30 mg/L of culture) in soluble form. We describe here a simple two-step purification protocol that produces reasonable quantities of homogeneous enzyme. In denaturing gel electrophoresis the enzyme behaved as a approximately 90 kDa protein. The native structure, determined by gel-filtration studies, appeared to be hexameric and its quaternary structure was salt (NaCl) dependent. In low-salt buffers (containing 50 mM NaCl), the enzyme eluted in a single activity peak at an elution volume that appeared to correlate with a possible hexameric structure. In higher salt buffer (containing greater than 150 mM NaCl), the enzyme eluted as smaller assemblies (monomer/dimer). The recombinant helicase A was able to hydrolyze ATP or dATP with equal efficiency. The ATPase activity of the enzyme was absolutely DNA-dependent. The nucleotidase activities were comparable to those of the native enzyme. Kinetic analysis of the ATPase activity demonstrated that the Km of the enzyme was approximately 90 microM and the rate of ATP hydrolysis was approximately 20 ATP s-1 molecule-1. DNA sequences containing pyrimidine stretches were more effective activators than those containing purine stretches. However, poly(dC) appeared to be the most effective activator of the ATPase activity. The ATPase activity was inhibited by salt (NaCl) above 50 mM with a half-maximal inhibition at approximately 110 mM. It is likely that the active state of helicase A is hexameric. The helicase activity of the recombinant enzyme was stimulated significantly by the yeast replication protein A (RPA) and to a lower extent by the single-stranded DNA binding protein of E. coli (SSB). The DNA helicase migrated on a DNA template in a 5' --> 3' direction. Helicase A appeared to share a number of enzymatic characteristics including directionality, stimulation by RPA/SSB, and quaternary structure (monomer-hexamer) dynamics that are common to known replicative helicases such as DnaB helicase and the SV40 T-antigen.

Stimulation of RTH1 nuclease of the yeast Saccharomyces cerevisiae by replication protein A.

The RTH1 nuclease is involved in the replication of chromosomal DNA as well as in the repair of DNA damage. Replication protein A (RPA) is also an integral part of the DNA replication and repair processes. We have investigated the roles(s) of RPA in the function of RTH1 nuclease, including its structure specific endonuclease activity. Initial in vitro studies, which employed a "flap" or a "pseudo Y" substrate containing a short 14 bp duplex region, showed the effect of RPA to be minimal or inhibitory. As RPA inhibition is unwarranted for a protein participating in the DNA replication process, we have further investigated the mechanism of such inhibition. Alternate flap and pseudo Y substrates with a long duplex region (50 bp) were prepared using M13mp19 ssDNA and synthetic oligonucleotides. Yeast RPA stimulated the endonuclease activity of RTH1 endonuclease with these substrates in a dose-dependent manner. Kinetic analysis suggested that yRPA exerted a bipartite effect on the nuclease reaction: (i) the "load time" of RTH1 nuclease onto the DNA substrate decreased from approximately 5 to 2 min in the presence of RPA, and (ii) following initiation of the nuclease reaction, the initial rate of the reaction increased 10-fold in the presence of yRPA. Further analysis of the interaction of RPA with various endonuclease substrates indicated that RPA has a weak helix destabilizing effect and could melt small, 14 bp, regions of duplex DNA. RTH1 endonuclease cleaves the DNA strand at the junction of single- and double-stranded DNA; consequently, the observed inhibition with small duplex substrates was likely due to duplex melting. Our studies also demonstrated that RPA stimulated the RNase H activity of RTH1 nuclease significantly. In both instances (RTH1 endonuclease and RNase H), the stimulation may involve a specific interaction of RPA with the RTH1 nuclease rather than a structural positioning of the DNA substrate by RPA.

Purification and characterization of the DNA polymerase alpha associated exonuclease: the RTH1 gene product.

We report here the purification and mechanistic characterization of a 5'-3' exonuclease associated with DNA polymerase alpha from the yeast Saccharomyces cerevisiae. Earlier, we identified a 5' --> 3' exonuclease activity that copurified with yeast DNA polymerase alpha-primase in a multiprotein complex [Biswas, E. E., et al. (1993) Biochemistry, 32, 3020-3027]. Peptide sequence analysis of the purified 47 kDa exonuclease was carried out, and the peptide sequence was found to be identical to the S. cerevisiae gene YKL510 encoded polypeptide, which is also known as yeast RAD2 homolog 1 or RTH1 nuclease. The native exonuclease also had strong flap endonuclease activity similar to that observed with RTH1 nuclease and homologous yeast (RAD2) and mammalian enzymes. During our studies, we have discovered certain unique features of the mechanism of action of the native RTH1 nuclease. Studies presented here indicated that the exonuclease had specific pause sites during its 5'-3' exonuclease nucleotide excision. These pause sites were easily detected with long (approximately 50 bp) oligonucleotide substrates during exonucleolytic excision by the formation of a discontinuous ladder of excision product. We have further analyzed the mechanism of generation of the pause sites, as they could occur through a number of different pathways. Alignment of the pause sites with the nucleotide sequence of the oligonucleotide substrate indicated that the pause sites were dependent on the nucleotide sequence. Our analysis revealed that RTH1 nuclease pauses predominantly at G:C rich sequences. With poly(dA):oligo(dT)50 as substrate, the exonucleolytic products formed a continuous ladder with no evidence of pausing. The G:C rich DNA sequences are thermodynamically more stable than the A:T rich sequences, which may be in part responsible for pausing of the RTH1 5' --> 3' exonuclease at these sites.

Overexpression and rapid purification of biologically active yeast proliferating cell nuclear antigen.

Yeast proliferating cell nuclear antigen (yPCNA) is a cell-cycle-regulated protein that has been shown to be required for the efficient elongation of primed DNA templates by DNA polymerase delta in vitro. We have expressed yPCNA to a high level (> or = 30% of the total cellular protein) with and without a six-residue histidine tag at its amino-terminus. Both forms of the recombinant protein were found to be biologically active and no significant differences were observed between the two forms. In this report we describe an efficient method of extraction of DNA binding proteins, such as yPCNA, overexpressed in Escherichia coli. The presence of a (His) delta tag on the polypeptide permitted rapid and high-yield single-step purification of the protein (approximately 60 mg of purified yPCNA per liter of induced cell culture) by immobilized metal affinity chromatography using an imidazole gradient elution procedure. The purified yPCNA was used to characterize the biological activity and tertiary structure of the protein. Chemical crosslinking and size-exclusion FPLC studies indicated that both forms of the protein have a trimeric-oligomeric structure in solution. Taken together these results indicate that both forms of the recombinant yPCNA were similar to the endogenous protein in their biochemical properties. The strategies presented here are designed to maximize the yield of recombinant protein and should prove useful to the purification of other recombinant DNA binding proteins.

Biochemical and genetic characterization of a replication protein A dependent DNA helicase from the yeast, Saccharomyces cerevisiae.

We have purified a DNA dependent ATPase/DNA helicase, DNA helicase B, from S. cerevisiae. Helicase B was a 129-kDa polypeptide. The ATPase activity of helicase B was strongly DNA dependent. The DNA helicase activity was stimulated by yeast replication protein A, indicating a probable function in DNA replication. Helicase B showed a 5'-->3' polarity of movement. Protein sequencing indicated that helicase B was identical to a hypothetical 127-kDa polypeptide encoded by yORF61, located 5' upstream of the BMH1 locus in chromosome V. The protein sequence contained a "type I ATP/GTP binding motif" and other helicase-like motifs and the expressed protein exhibited helicase activity. Thus, we concluded that yORF61 is the gene for helicase B and will be referred to as HCSB.

A low-affinity estrogen-binding site in pregnant rat uteri: analysis and partial purification.

We have identified a low-affinity (type II) estrogen-binding site (EBS) that is expressed at high levels during pregnancy in rat uteri. Although this activity was detectable in nonpregnant rat uteri, it was present in amounts (0.094 pmol/g of uteri) that were severalfold lower than the high-affinity type I estrogen receptor (0.57 pmol/g of uteri). During pregnancy, at 19-20 days of gestation, the low-affinity type II EBS became the major (> or = 88%) estrogen-binding site in rat uteri. The increase in the level of low-affinity EBS (7.9 pmol/g) in uteri was approximately 85-fold with an approximately 20-fold increase in the specific activity (0.39 pmol/mg) of this form, whereas the high-affinity form remained relatively unchanged. We report here a method of purification of type II EBS from pregnant rat uteri and present an analysis of its DNA and steroid-binding properties. Estradiol-binding studies and Scatchard analysis showed that the type II EBS had an apparent estradiol-binding affinity of > or = 24 nM. Gel filtration and SDS/PAGE analysis indicated that the type II EBS was a monomeric 73-kDa protein. The estradiol binding remained apparently uninhibited in the presence of a large excess of tamoxifen, nafoxidine, or dihydrotestosterone. Estradiol, diethylstilbestrol, and quercitin (a type II EBS-specific inhibitor) competed efficiently. The purified low-affinity EBS did not have sequence-specific DNA-binding activity with the estrogen-responsive element, which indicated that it differs in function from the type I estrogen receptor.

Structure and function of Escherichia coli DnaB protein: role of the N-terminal domain in helicase activity.

We have analyzed the contributions of specific domains of DnaB helicase to its quaternary structure and multienzyme activities. Highly purified tryptic fragments containing various domains of DnaB helicase were prepared. Fragment I lacks 14 amino acid (aa) residues from the N-terminal of DnaB helicase. Fragments II and III are 33-kDa C-terminal and 12-kDa N-terminal polypeptides, respectively, of fragment I. The single-stranded DNA-dependent ATPase and DNA helicase activities of DnaB helicase and its fragments were examined in detail. The ATPase activities of native DnaB helicase and fragment I were comparable; however, the ATPase activity of fragment II was somewhat diminished. Unlike the ATPase activity, the DNA helicase activity was totally abolished in fragment II and was not complemented by the addition of equimolar fragment III. Consequently, the N-terminal 17-kDa domain appeared to have an indispensable role in the DNA helicase action, but not in other enzymatic activities. Fragment I had a hexameric structure similar to that observed with DnaB helicase in both size exclusion HPLC (SE-HPLC) and chemical cross-linking studies. SE-HPLC analysis indicated that fragment II had an apparent hexameric form. However, a detailed chemical cross-linking analysis showed that it formed stable dimers but the formation of a stable hexamer was severely impaired. Thus, the N-terminal domain appeared to have a strong influence on the hexamer formation.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA helicase associated with DNA polymerase alpha: isolation by a modified immunoaffinity chromatography.

We have developed a novel immunoaffinity method for isolating a DNA polymerase alpha-associated DNA helicase from the yeast Saccharomyces cerevisiae. Earlier we have reported the characterization of a DNA helicase activity associated with the multiprotein DNA polymerase alpha complex from yeast [Biswas, E. E., Ewing, C. M., & Biswas, S. B. (1993) Biochemistry 32, 3030-3027]. We report here the isolation of the DNA helicase from the DNA polymerase alpha (pol alpha) complex bound to an anti-pol alpha immunoaffinity matrix. The DNA helicase activity eluted at approximately 0.35 M NaCl concentration. The eluted ATPase/helicase peak was further purified by size-exclusion high-performance liquid chromatography (HPLC). At low ionic strength (50 mM NaCl), it remained associated with other proteins and eluted as a large polypeptide complex. At high ionic strength (500 mM NaCl), the helicase dissociated, and the eluted ATPase/helicase fraction contained 90-, 60-, and 50-kDa polypeptides. Photoaffinity cross-linking of helicase with ATP during the isolation process demonstrated a 90-kDa polypeptide to be the likely ATP binding component of the helicase protein. The DNA helicase has single-stranded DNA (ssDNA)-stimulated ATPase and dATPase activities. The ATPase activity was stimulated by yeast replication protein A (RPA). The DNA helicase activity was stimulated by Escherichia coli ssDNA binding protein and RPA. The DNA helicase migrated on a DNA template in the 5'-->3' direction which is also the overall direction of migration of pol alpha on the lagging strand of the replication fork.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the DNA-dependent ATPase and a DNA unwinding activity associated with the yeast DNA polymerase alpha complex.

We have analyzed the ATPase and dATPase activities associated with the yeast DNA polymerase alpha complex. The ATPase/dATPase was primarily a single-stranded DNA-dependent ATPase. Analysis of the stimulatory effect of a large number of DNA substrates demonstrated that polynucleotides longer than 60 nucleotides (nts) had the maximal effect. The stimulation by oligonucleotides smaller than 60 nts, in general, decreased proportionally with decreased length of the oligomer. Poly- or oligopyrimidines were twice as stimulatory as the poly- or oligopurines of the same length. In addition to DNA, replication protein A (RP-A), a single-stranded DNA (ssDNA) binding protein, also stimulated the ATPase activity. Photo-cross-linking of the ATP binding component of the pol alpha complex to [alpha-32P]ATP at 0 degree C resulted in the exclusive labeling of a 90-kDa polypeptide. The labeling was inhibited by ATP and dATP but not by any other ribo- or deoxynucleotides, which suggest that the 90-kDa polypeptide is specific for ATP/dATP binding and possibly the active site for the ATPase/dATPase. We have also reported here a novel DNA unwinding activity associated with the multiprotein complex of DNA polymerase alpha. The complex was able to unwind M13mp19 ssDNA hybridized to an oligonucleotide (17-60 nucleotides long) with a protruding 3'-terminus. Regardless of the size of the duplex, the DNA unwinding was significantly stimulated by RP-A, while RP-A itself did not have any DNA unwinding activity. Consequently, it appeared that the DNA polymerase alpha complex possessed a putative RP-A-dependent helicase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of a yeast DNA polymerase alpha complex with associated primase, 5'-->3' exonuclease, and DNA-dependent ATPase activities.

We have purified a multimeric form of yeast DNA polymerase alpha with DNA polymerase, primase, 5'-->3' exonuclease, and single-stranded (ss) DNA-dependent ATPase activities to near-homogeneity. The molecular mass of complex was 650 kDa with subunits ranging in sizes from 30 to 180 kDa. The alpha-subunit of the complex could be detected by DNA polymerase alpha antibody. No cross-reactivity of polypeptides within the complex was observed with antibodies directed against polymerase delta or epsilon. The multimeric polymerase alpha could be selectively inhibited by p-n-butylphenyl-dGTP (I50 of approximately 0.2 microM), p-n-butylanilino-dATP (I50 of 1.3 microM), and aphidicolin (I50 of 2.5 micrograms/mL). The complex synthesized RNA primers on various ssDNA templates and rapidly elongated these primers into nascent DNA fragments in the presence of required deoxynucleotides. It has a strong 5'-->3' exonuclease activity. In addition, the complex hydrolyzed both ATP and dATP in a ssDNA-dependent manner. Thus, the multiprotein complex of DNA polymerase alpha had multiple activities (primase, polymerase, and ATPase) which could act concertedly to synthesize primers and elongate the primers to nascent DNA fragments in the lagging strand of the fork.

Molecular cloning of a gene encoding an ARS binding factor from the yeast Saccharomyces cerevisiae.

We report the isolation of the gene for origin binding factor 1 (OBF1) from the yeast Saccharomyces cerevisiae by screening a yeast genomic DNA library in lambda gt11 with an ARS-specific oligonucleotide probe. One recombinant encoded a fusion protein of approximately 180 kDa that bound ARS-specific oligonucleotide probes in vitro. The restriction map of this gene was determined after isolation of the complete gene by screening a yeast genomic DNA library in YEp24. Characterization of the gene for OBF1 by pulsed-field gel electrophoresis and Northern and Southern blot analyses demonstrated that (i) the gene is located in chromosome IV, (ii) the gene is a single-copy gene, (iii) the mRNA is approximately 3.8 kilobases, which could code for an approximately 130-kDa polypeptide, consistent with the reported size of OBF1. An antibody, affinity-purified using the lysogen-encoded fusion protein, specifically detected an approximately 130-kDa polypeptide in yeast extract. The isolation of the gene for OBF1 should allow further analysis of the mechanism of action of this protein in vitro and in vivo.

ARS binding factor I of the yeast Saccharomyces cerevisiae binds to sequences in telomeric and nontelomeric autonomously replicating sequences.

We have analyzed various autonomously replicating sequences (ARSs) in yeast nuclear extract with ARS-specific synthetic oligonucleotides. The EI oligonucleotide sequence, which is derived from HMRE-ARS, and the F1 oligonucleotide sequence, which is derived from telomeric ARS120, appeared to bind to the same cellular factor with high specificity. In addition, each of these oligonucleotides was a competitive inhibitor of the binding of the other. Binding of the ARS binding factor (ABF) to either of these oligonucleotides was inhibited strongly by plasmids containing ARS1 and telomeric TF1-ARS. DNase I footprinting analyses with yeast nuclear extract showed that EI and F1 oligonucleotides eliminated protection of the binding site of ARS binding factor I (ABFI) in domain B of ARS1. Sequence analyses of various telomeric (ARS120 and TF1-ARS) and nontelomeric ARSs (ARS1 and HMRE-ARS) showed the presence of consensus ABFI binding sites in the protein binding domains of all of these ARSs. Consequently, the ABFI and ABFI-like factors bind to these domain B-like sequences in a wide spectrum of ARSs, both telomeric and nontelomeric.

Replication of single-stranded DNA templates by primase-polymerase complexes of the yeast, Saccharomyces cerevisiae.

A partially purified primase-polymerase complex from the yeast, Saccharomyces cerevisiae, was capable of replicating a single stranded circular phage DNA into a replicative form with high efficiency. The primase-polymerase complex exhibited primase activity and polymerase activity on singly primed circular ssDNA as well as on gapped DNA. In addition, it was able to replicate an unprimed, single-stranded, circular phage DNA through a coupled primase-polymerase action. On Biogel A-O.5m filtration the primase-polymerase activities appeared in the void volume, demonstrating a mass of greater than 500 kilodaltons. Primase and various primase-polymerase complexes synthesized unique primers on single stranded DNA templates and the size distribution of primers was dependent on the structure of the DNA and the nature of the primase-polymerase assembly.

Yeast DNA primase is encoded by a 59-kilodalton polypeptide: purification and immunochemical characterization.

The DNA primase from the yeast Saccharomyces cerevisiae has been purified 9200-fold to homogeneity. The yeast DNA primase is a monomeric protein of molecular weight 59,000, and under conditions described in this report, it is stable at 4 or -80 degrees C. The primase does not bind to DEAE-cellulose, is not inhibited by a high concentration of alpha-amanitin (4 mg/mL), and is capable of synthesizing small (up to 15 nucleotides in length) ribo or ribo-deoxy mixed initiator RNA primers. The primer synthesis is stimulated by ATP; however, other ribonucleotides could be replaced by deoxynucleotides without any measurable effect on the overall DNA synthesis. Thus, the purified primase is distinct from the RNA polymerases of S. cerevisiae. Immunoblot analysis of the polypeptides in a crude cell extract using a mouse polyclonal antibody prepared against the highly purified primase indicates that the 59-kilodalton polypeptide is the native form and not a degraded form of a larger polypeptide; however, primase is degraded rapidly to smaller polypeptides by yeast proteases especially in the absence of protease inhibitors.