Biochemical Characterization of Adeno-Associated Virus Rep68 DNA Helicase and ATPase Activities

The adeno-associated virus (AAV) nonstructural proteins Rep68 and Rep78 are site-specific DNA binding proteins, ATP-dependent site-specific endonucleases, helicases, and ATPases. These biochemical activities are required for viral DNA replication and control of viral gene expression. In this study, we characterized the biochemical properties of the helicase and ATPase activities of homogeneously pure Rep68. The enzyme exists as a monomer in solution at the concentrations used in this study (<380 nM), as judged by its mobility in sucrose density gradients. Using a primed single-stranded (ss) circular M13 substrate, the helicase activity had an optimum pH of 7 to 7.5, an optimum temperature of 45°C, and an optimal divalent-cation concentration of 5 mM MgCl₂. Several nucleoside triphosphates could serve as cofactors for Rep68 helicase activity, and the order of preference was ATP = GTP > CTP = dATP > UTP > dGTP. The K_m values for ATP in both the DNA helicase reaction and the site-specific trs endonuclease reaction were essentially the same, approximately 180 μM. Both reactions were sigmoidal with respect to ATP concentration, suggesting that a dimer or higher-order multimer of Rep68 is necessary for both DNA helicase activity and terminal resolution site (trs) nicking activity. Furthermore, when the enzyme itself was titrated in the trs endonuclease and ATPase reactions, both activities were second order with respect to enzyme concentration. This suggests that a dimer of Rep68 is the active form for both the ATPase and nicking activities. In contrast, DNA helicase activity was linear with respect to enzyme concentration. When bound to ssDNA, the enzyme unwound the DNA in the 3′-to-5′ direction. DNA unwinding occurred at a rate of approximately 345 bp per min per monomeric enzyme molecule. The ATP turnover rate was approximately 30 to 50 ATP molecules per min per enzyme molecule. Surprisingly, the presence of DNA was not required for ATPase activity. We estimated that Rep translocates processively for more than 1,300 bases before dissociating from its substrate in the absence of any accessory proteins. DNA helicase activity was not significantly stimulated by substrates that have the structure of a replication fork and contain either a 5′ or 3′ tail. Rep68 binds only to ssDNA, as judged by inhibition of the DNA helicase reaction with ss or double-stranded (ds) DNA. Consistent with this observation, no helicase activity was detected on blunt-ended ds oligonucleotide substrates unless they also contained an ss 3′ tail. However, if a blunt-ended ds oligonucleotide contained the 22-bp Rep binding element sequence, Rep68 was capable of unwinding the substrate. This means that Rep68 can function both as a conventional helicase for strand displacement synthesis and as a terminal-repeat-unwinding protein which catalyzes the conversion of a duplex end to a hairpin primer. Thus, the properties of the Rep DNA helicase activity suggest that Rep is involved in all three of the key steps in AAV DNA replication: terminal resolution, reinitiation, and strand displacement.     

Identification and purification of a protein that stimulates the helicase activity of the Escherichia coli Rep protein

A polypeptide (Mr = 15,000) has been purified from Escherichia coli cell extracts that significantly stimulates the duplex DNA unwinding reaction catalyzed by E. coli Rep protein. The Rep helicase unwinding reaction was stimulated by as much as 20-fold, upon addition of the stimulatory protein, using either a 71-base pair or a 343-base pair partial duplex DNA molecule as a substrate. The purified Rep helicase stimulatory protein (RHSP) had no intrinsic helicase activity or ATP hydrolysis activity and did not stimulate the single-stranded DNA-dependent ATP hydrolysis reaction catalyzed by Rep protein. It is likely that RHSP stimulates the Rep helicase unwinding reaction by stoichiometric binding to single-stranded DNA. However, a specific interaction between Rep protein and RHSP cannot be ruled out, since RHSP did not stimulate the duplex DNA unwinding reactions catalyzed by E. coli helicase I or the recently discovered 75-kDa helicase. RHSP did stimulate the duplex DNA unwinding reaction catalyzed by E. coli helicase II. The identification and subsequent purification of RHSP from cell extracts demonstrates the feasibility of using direct helicase assays to purify stimulatory proteins.     

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.     

Localization of the sites of ADP-ribosylation and GTP binding in the eukaryotic elongation factor EF-2

Tryptic cleavage of EF-2, molecular mass 93 kDa, produced an 82-kDa polypeptide and a 10-kDa fragment, which was further degraded. By a slower reaction the 82-kDa polypeptide was gradually split into a 48-kDa and a 34-kDa fragment. Similarly, treatment with chymotrypsin resulted in the formation of an 82-kDa polypeptide and a small fragment. In contrast to the tryptic 82-kDa polypeptide the corresponding chymotryptic cleavage product was relatively resistant to further attack. The degradation of the 82-kDa polypeptide with either trypsin or chymotrypsin was facilitated by the presence of guanosine nucleotides, indicating a conformational shift in native EF-2 upon nucleotide binding. No effect was observed in the presence of ATP, indicating that the effect was specific for guanosine nucleotides. After affinity labelling of native EF-2 with oxidized [3H]GTP and subsequent trypsin treatment the radioactivity was recovered in the 48-kDa polypeptide showing that the GTP-binding site was located within this part of the factor. Correspondingly, tryptic degradation of EF-2 labelled with [14C]NAD+ in the presence of diphtheria toxin showed that the site of ADP-ribosylation was within the 34-kDa polypeptide. By cleavage with the tryptophan-specific reagent N-chlorosuccinimide the site of ADP-ribosylation could be located at a distance of 40-60 kDa from the GTP-binding site and about 4-11 kDa from the nearest terminus.     

Limited tryptic digestion of Acanthamoeba myosin IA abolishes regulation of actin-activated ATPase activity by heavy chain phosphorylation

Acanthamoeba myosin IA is a globular protein composed of a 140-kDa heavy chain and a 17-kDa light chain. It expresses high actin-activated Mg2+-ATPase activity when one serine on the heavy chain is phosphorylated. We previously showed that chymotrypsin cleaves the heavy chain into a COOH-terminal 27-kDa peptide that can bind to F-actin but has no ATPase activity and a complex containing the NH2-terminal 112-kDa peptide and the light chain. The complex also binds F-actin and has full actin-activated Mg2+-ATPase activity when the regulatory site is phosphorylated. We have now localized the ATP binding site to within 27 kDa of the NH2 terminus and the regulatory phosphorylatable serine to a 20-kDa region between 38 and 58 kDa of the NH2 terminus. Under controlled conditions, trypsin cleaves the heavy chain at two sites, 38 and 112 kDa from the NH2 terminus, producing a COOH-terminal 27-kDa peptide similar to that produced by chymotrypsin and a complex consisting of an NH2-terminal kDa peptide, a central 74-kDa peptide, and the light chain. This complex is similar to the chymotryptic complex but for the cleavage which separates the 38- and 74-kDa peptides. The tryptic complex has full (K+, EDTA)-ATPase activity (the catalytic site is functional) and normal ATP-sensitive actin-binding properties. However, the actin-activated Mg2+-ATPase activity and the F-actin-binding characteristics of the tryptic complex are no longer sensitive to phosphorylation of the regulatory serine. Therefore, cleavage between the phosphorylation site and the ATP-binding site inhibits the effects of phosphorylation on actin binding and actin-activated Mg2+-ATPase activity without abolishing the interactions between the ATP- and actin-binding sites.     

Factors Affecting the Terminal Resolution Site Endonuclease, Helicase, and ATPase Activities of Adeno-Associated Virus Type 2 Rep Proteins

The Rep68 and Rep78 proteins (Rep68/78) of adeno-associated virus type 2 (AAV) are critical for AAV replication and site-specific integration. They bind specifically to the AAV inverted terminal repeats (ITRs) and possess ATPase, helicase, and strand-specific/site-specific endonuclease activities. In the present study, we further characterized the AAV Rep68/78 helicase, ATPase, and endonuclease activities by using a maltose binding protein-Rep68 fusion (MBP-Rep68Delta) produced in Escherichia coli cells and Rep78 produced in vitro in a rabbit reticulocyte lysate system. We found that the minimal length of single-stranded DNA capable of stimulating the ATPase activity of MBP-Rep68Delta is 100 to 200 bases. The degree of stimulation correlated positively with the length of single-stranded DNA added to the reaction mixture. We then determined the ATP concentration needed for optimal MBP-Rep68Delta helicase activity and showed that the helicase is active over a wide range of ATP concentrations. We determined the directionality of MBP-Rep68Delta helicase activity and found that it appears to move in a 3' to 5' direction, which is consistent with a model in which AAV Rep68/78 participates in AAV DNA replication by unwinding DNA ahead of a cellular DNA polymerase. In this report, we also demonstrate that single-stranded DNA is capable of inhibiting the MBP-Rep68Delta or Rep78 endonuclease activity greater than 10-fold. In addition, we show that removal of the secondary Rep68/78 binding site, which is found only in the hairpin form of the AAV ITR, causes a three- to eightfold reduction in the ability of the ITR to be used as a substrate for the Rep78 or MBP-Rep68Delta endonuclease activity. This suggests that contact between Rep68/78 and this secondary element may play an important role in the Rep-mediated endonuclease activity.     

Rep protein as a helicase in an active, isolatable replication fork of duplex phi X174 DNA

Rep protein as a helicase combines its actions with those of gene A protein and single-stranded DNA binding protein to separate the strands of phi X174 duplex DNA and thereby can generate and advance a replication fork (Scott, J. F., Eisenberg, S., Bertsch, L. L., and Kornberg, A. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 193-197). Tritium-labeled rep protein is bound in an active gene A protein. phi X174 closed circular duplex supercoiled DNA complex in a 1:1 ratio. Catalytic separation of the strands of the duplex by rep protein, as measured by incorporation of tritium-labeled single-stranded DNA binding protein, requires ATP at a Km value of 8 microM, and hydrolyzes two molecules of ATP for every base pair melted. When coupled to replication in the synthesis of single-strand viral circles, a "looped" rolling-circle intermediate is formed that can be isolated in an active form containing gene A protein, rep protein, single-stranded DNA binding protein, and DNA polymerase III holoenzyme. Unlike the binding of rep protein to single-stranded DNA, where its ATPase activity is distributive, binding to the replicating fork is not affected by ATP, further suggesting a processive action linked to gene A protein. Limited tryptic hydrolysis of rep protein abolishes its replicative activity without affecting significantly its binding of ATP and its ATPase action on single-stranded DNA. These results augment earlier findings by describing the larger role of rep proteins as a helicase, linked in a complex ith other proteins, at the replication fork of a duplex DNA.     

Limited proteolysis as a probe of the conformation and nucleic acid binding regions of nucleolin.

Nucleolin, also called protein C23, is a RNA-associated protein implicated in the early stages of ribosome assembly. To study the general conformation and map the nucleic acid binding regions, rat nucleolin was subjected to limited proteolysis using trypsin and chymotrypsin in the presence or absence of poly(G). The cleavage sites were classified according to their locations in the three putative domains: the highly polar amino-terminal domain, the central nucleic acid binding domain, which contains four 90-residue repeats, and the carboxyl-terminal domain, which is rich is glycine, dimethylarginine, and phenylalanine. The most labile sites were found in basic segments of the amino-terminal domain. This region was stabilized by Mg2+. At low enzyme concentrations, cleavage by trypsin or chymotrypsin in the amino-terminal domain was enhanced by poly(G). Trypsin produced a relatively stable 48-kDa fragment containing the central and carboxyl-terminal domains. The enhanced cleavage suggests that binding of nucleic acid by the central domain alters the conformation of the amino-terminal domain, exposing sites to proteolytic cleavage. At moderate enzyme concentrations, the 48-kDa fragment was protected by poly(G) against tryptic digestion. At the highest enzyme concentrations, both enzymes cleaved near the boundaries between repeats 2, 3, and 4 with some sites protected by poly(G), suggesting that the repeats themselves form compact units. The carboxyl-terminal domain was resistant to trypsin but was cleaved by chymotrypsin either in the presence or in the absence of poly(G), indicating exposure of some phenylalanines in this region. These studies provide a general picture of the topology of nucleolin and suggest that the nucleic acid binding region communicates with the amino-terminal domain.     

A maltose-binding protein/adeno-associated virus Rep68 fusion protein has DNA-RNA helicase and ATPase activities.

The adeno-associated virus type 2 (AAV) Rep68 protein produced in Escherichia coli as a fusion protein with maltose-binding protein (MBP-Rep68 delta) has previously been shown to possess DNA-DNA helicase activity, as does the purified wild-type Rep68. In the present study, we demonstrate that MBP-Rep68 delta also catalyzes the unwinding of a DNA-RNA hybrid. MBP-Rep68 delta-mediated DNA-RNA helicase activity required ATP hydrolysis and the presence of Mg2+ ions and was inhibited by high ionic strength. The efficiency of the DNA-RNA helicase activity of MBP-Rep68 delta was comparable to its DNA-DNA helicase activity. However, MBP-Rep68 delta lacked the ability to unwind a blunt-ended DNA-RNA substrate and RNA-RNA duplexes. We have also demonstrated that MBP-Rep68 delta has ATPase activity which is enhanced by the presence of single-stranded DNA but not by RNA. The MBP-Rep68 delta NTP mutant protein, which has a lysine-to-histidine substitution at amino acid 340 in the putative nucleoside triphosphate-binding site of Rep68, not only lacks DNA-RNA helicase and ATPase activities but also inhibits the helicase activity of MBP-Rep68 delta. DNA-RNA helicase activity of Rep proteins might play a pivotal role in the regulation of AAV gene expression by AAV Rep proteins.     

Purification and characterization of a new DNA-dependent ATPase with helicase activity from Escherichia coli

A previously unreported single-stranded DNA-dependent nucleoside 5'-triphosphatase with DNA unwinding activity has been purified from extracts of Escherichia coli lacking the F factor. Fractions of the purified enzyme contain a major polypeptide of Mr = 75,000 which contains the active site(s) for both ATP hydrolysis and helicase activity. This is consistent with the results of gel filtration chromatography which indicate a native molecular mass of 75 kDa. The 75-kDa helicase has a preference for ATP (dATP) as a substrate in the hydrolysis reaction and requires the presence of a single-stranded DNA cofactor. The helicase reaction catalyzed by the enzyme has been characterized using an in vitro strand displacement assay. The 75-kDa helicase displaces a 71-nucleotide DNA fragment in an enzyme concentration-dependent and time-dependent reaction. The helicase reaction depends on the presence of a hydrolyzable nucleoside 5'-triphosphate (NTP) suggesting that NTP hydrolysis is required for the unwinding activity. In addition, the enzyme can displace a 343-nucleotide DNA fragment albeit less efficiently. The direction of the unwinding reaction is 3' to 5' with respect to the strand of DNA on which the enzyme is bound. The molecular size of this helicase and the direction of the unwinding reaction are similar to both helicase II and Rep protein. However, the 75-kDa helicase has been shown to be distinct from both helicase II and Rep protein using immunological, physical, and genetic criteria. The discovery of a new helicase brings the total number of helicases found in E. coli cell extracts (lacking F factor) to five.     

Biochemical characterization of the DNA helicase activity of the escherichia coli RecQ helicase

We demonstrate that RecQ helicase from Escherichia coli is a catalytic helicase whose activity depends on the concentration of ATP, free magnesium ion, and single-stranded DNA-binding (SSB) protein. Helicase activity is cooperative in ATP concentration, with an apparent S(0.5) value for ATP of 200 microm and a Hill coefficient of 3.3 +/- 0.3. Therefore, RecQ helicase utilizes multiple, interacting ATP-binding sites to mediate double-stranded DNA (dsDNA) unwinding, implicating a multimer of at least three subunits as the active unwinding species. Unwinding activity is independent of dsDNA ends, indicating that RecQ helicase can unwind from both internal regions and ends of dsDNA. The K(M) for dsDNA is 0.5-0.9 microm base pairs; the k(cat) for DNA unwinding is 2.3-2.7 base pairs/s/monomer of RecQ helicase; and unexpectedly, helicase activity is optimal at a free magnesium ion concentration of 0.05 mm. Omitting Escherichia coli SSB protein lowers the rate and extent of dsDNA unwinding, suggesting that RecQ helicase associates with the single-stranded DNA (ssDNA) product. In agreement, the ssDNA-dependent ATPase activity is reduced in proportion to the SSB protein concentration; in its absence, ATPase activity saturates at six nucleotides/RecQ helicase monomer and yields a k(cat) of 24 s(-1). Thus, we conclude that SSB protein stimulates RecQ helicase-mediated unwinding by both trapping the separated ssDNA strands after unwinding and preventing the formation of non-productive enzyme-ssDNA complexes.     

Subdomain organization and catalytic residues of the F factor TraI relaxase domain

TraI from conjugative plasmid F factor is both a "relaxase" that sequence-specifically binds and cleaves single-stranded DNA (ssDNA) and a helicase that unwinds the plasmid during transfer. Using limited proteolysis of a TraI fragment, we generated a 36-kDa fragment (TraI36) retaining TraI ssDNA binding specificity and relaxase activity but lacking the ssDNA-dependent ATPase activity of the helicase. Further proteolytic digestion of TraI36 generates stable N-terminal 26-kDa (TraI26) and C-terminal 7-kDa fragments. Both TraI36 and TraI26 are stably folded and unfold in a highly cooperative manner, but TraI26 lacks affinity for ssDNA. Mutational analysis of TraI36 indicates that N-terminal residues Tyr(16) and Tyr(17) are required for efficient ssDNA cleavage but not for high-affinity ssDNA binding. Although the TraI36 N-terminus provides the relaxase catalytic residues, both N- and C-terminal structural domains participate in binding, suggesting that both domains combine to form the TraI relaxase active site.     

The amino acid linker between the endonuclease and helicase domains of adeno-associated virus type 5 Rep plays a critical role in DNA-dependent oligomerization

The adeno-associated virus (AAV) genome encodes four Rep proteins, all of which contain an SF3 helicase domain. The larger Rep proteins, Rep78 and Rep68, are required for viral replication, whereas Rep40 and Rep52 are needed to package AAV genomes into preformed capsids; these smaller proteins are missing the site-specific DNA-binding and endonuclease domain found in Rep68/78. Other viral SF3 helicases, such as the simian virus 40 large T antigen and the papillomavirus E1 protein, are active as hexameric assemblies. However, Rep40 and Rep52 have not been observed to form stable oligomers on their own or with DNA, suggesting that important determinants of helicase multimerization lie outside the helicase domain. Here, we report that when the 23-residue linker that connects the endonuclease and helicase domains is appended to the adeno-associated virus type 5 (AAV5) helicase domain, the resulting protein forms discrete complexes on DNA consistent with single or double hexamers. The formation of these complexes does not require the Rep binding site sequence, nor is it nucleotide dependent. These complexes have stimulated ATPase and helicase activities relative to the helicase domain alone, indicating that they are catalytically relevant, a result supported by negative-stain electron microscopy images of hexameric rings. Similarly, the addition of the linker region to the AAV5 Rep endonuclease domain also confers on it the ability to bind and multimerize on nonspecific double-stranded DNA. We conclude that the linker is likely a key contributor to Rep68/78 DNA-dependent oligomerization and may play an important role in mediating Rep68/78's conversion from site-specific DNA binding to nonspecific DNA unwinding.     

Limited proteolysis of yeast elongation factor 3. Sequence and location of the subdomains

Elongation factor 3 (EF-3) is an ATPase essential for polypeptide chain synthesis in a variety of yeasts and fungi. We used limited proteolysis to study the organization of the subdomains of EF-3. Trypsinolysis of EF-3 at 30 degrees C resulted in the formation of three fragments with estimated molecular masses of 90, 70, and 50 kDa. Yeast ribosomes protected EF-3 and the large fragments from further degradation. ATP exposed a new tryptic cleavage site and stabilized the 70- and 50-kDa fragments. The conformation of EF-3 as measured by fluorescence spectroscopy did not change upon ATP binding. Poly(G) stimulated proteolysis and quenched the intrinsic fluorescence of EF-3. Using gel mobility shift, we demonstrated a direct interaction between EF-3 and tRNA. Neither tRNA nor rRNA altered the tryptic cleavage pattern. The proteolytic products were sequenced by mass spectrometric analysis. EF-3 is blocked NH(2)-terminally by an acetylated serine. The 90-, 70-, and 50-kDa fragments are also blocked NH(2)-terminally, confirming their origin. The 50-kDa fragment (Ser(2)-Lys(443)) is the most stable domain in EF-3 with no known function. The 70-kDa fragment (Ser(2)-Lys(668)) containing the first nucleotide-binding sequence motif forms the core ATP binding subdomain within the 90-kDa domain. The primary ribosome binding site is located near the loosely structured carboxyl-terminal end.     

Characterization of the catalytic subunit of an anion pump

The ArsA protein, the 63-kDa catalytic subunit of an oxyanion-translocating ATPase, was purified by successive chromatography using Q-Sepharose, red agarose, and phenyl-Sepharose to a specific activity in excess of 1 mumol of ATP hydrolyzed per min per mg of protein. ATPase activity was dependent on the presence of the oxyanionic substrates. Inhibitors of other classes of ion-translocating ATPases had no effect on ArsA ATPase activity, including N,N'-dicyclohexyl-carbodiimide, azide, vanadate, and nitrate. The apparent Km for ATP was determined to be 0.13 mM. The optimal pH range for ATP hydrolysis was 7.5 to 7.8. ATPase activity required Mg2+ at a molar ratio of 2 ATP:1 Mg2+. Limited proteolysis by trypsin was used to study conformational changes produced upon binding of substrates to the ArsA protein. In the absence of substrates, the ArsA protein was rapidly cleaved by trypsin to a major product of 30 kDa. ATP was partially protected from trypsin digestion, while the anionic substrate antimonite alone had no effect on proteolysis. Combination of the two substrates nearly completely protected the ArsA protein from proteolysis. Proteolytic cleavage correlated with loss of anion-stimulated ATPase activity and substrate protection from cleavage correlated with retention of activity. These results demonstrate that ATP and antimonite together produce a conformational change which is different from that of the ArsA protein in the presence of either substrate alone and suggest interaction between the oxyanion and ATP binding sites.     

Partial purification of adeno-associated virus Rep78, Rep52, and Rep40 and their biochemical characterization.

We have used differential cell extraction and conventional chromatography to separate and partially purify the four adeno-associated virus (AAV) nonstructural proteins Rep78, Rep68, Rep52, and Rep40. In the cytoplasmic extracts Rep52 and Rep40 were present in greater abundance than Rep68 and Rep78, with Rep78 being the least abundant. In nuclear extracts the four Rep proteins were approximately equal in abundance. Regardless of the subcellular fraction examined, three of the Rep proteins (Rep78, Rep68, and Rep40) consisted of two protein species with slightly different mobilities during polyacrylamide gel electrophoresis. In contrast, Rep52 consisted of only one protein species. Both Rep78 and Rep68 were capable of binding efficiently to AAV terminal hairpin DNA substrates, but we could not detect site-specific DNA binding by Rep52 and Rep40. Like Rep68, Rep78 had both an ATP-dependent trs endonuclease and a DNA helicase activity. Both Rep78 and Rep68 cut the terminal AAV sequence at the same site (nucleotide 124). The binding, trs endonuclease, and DNA helicase activities comigrated during sucrose density gradient centrifugation with a mobility expected for a monomer of the protein, suggesting that the three biochemical activities were intrinsic properties of the larger Rep proteins. The chromatographic behavior and the DNA-binding properties of the four Rep proteins identified at least two domains within the rep coding region, an exposed hydrophobic domain within the C-terminal end (amino acids 578 to 621) and a region within the N terminus (amino acids 1 to 214) which was necessary for binding to the terminal repeat sequence. No site-specific nuclease activity was seen in the presence of nucleotide analogs ATP-gamma-S or AMP-PNP, suggesting that ATP hydrolysis was required for the endonuclease reaction. Furthermore, although ATP was the only cofactor which would support the trs endonuclease activity of Rep78, Rep68 nuclease activity was seen in the presence of several other nucleotide cofactors, including CTP, GTP, and UTP.     

Characterization of structural domains of the human epidermal growth factor receptor obtained by partial proteolysis

Partial cleavage with trypsin has been used to study the structure of the epidermal growth factor (EGF) receptor purified from human carcinoma cells. Following affinity labeling of the receptor with 125I-EGF or the ATP analogue 5'-p-fluorosulfonyl benzoyl[14C]adenosine, metabolic labeling with [35S]methionine, [3H]glucosamine, or [32P]orthophosphate, or in vitro autophosphorylation with [gamma-32P]ATP, tryptic cleavage defines the following three regions of the 180-kDa receptor protein: 1) a 125-kDa trypsin-resistant domain which contains sites of glycosylation, EGF binding, and an EGF-specific threonine phosphorylation site; 2) an adjacent 40-kDa fragment which contains serine and threonine phosphorylation sites and is further cleaved to a 30-kDa trypsin-resistant domain; and 3) a terminal 15-kDa portion of the receptor that contains the sites of tyrosine phosphorylation and is degraded to small fragments in the presence of trypsin. Both the 125- and 40-kDa regions of the EGF receptor appear to be required for receptor-associated protein kinase activity since separation of these regions by tryptic cleavage abolishes this activity, and both regions are specifically labeled with an ATP affinity analogue, suggesting that both are involved in ATP binding. Additional 63- and 48-kDa phosphorylated fragments are generated upon trypsin treatment of EGF receptor from EGF-treated cells. The potential usefulness of partial tryptic cleavage in studying the EGF receptor and the possible biological function of the 30-kDa trypsin-resistant fragment of the receptor are discussed.     

A biochemical characterization of the adeno-associated virus Rep40 helicase

The human adeno-associated virus (AAV) has generated much enthusiasm as a transfer vector for human gene therapy. Although clinical gene therapy trials have been initiated using AAV vectors, much remains to be learned regarding the basic mechanisms of virus replication, gene expression, and virion assembly. AAV encodes four nonstructural, or replication (Rep), proteins. The Rep78 and Rep68 proteins regulate viral DNA replication, chromosomal integration, and gene expression. The Rep52 and Rep40 proteins mediate virus assembly. To better understand Rep protein function, we have expressed the Rep40 protein in Escherichia coli and purified it to near homogeneity. Like the other Rep proteins, Rep40 possesses helicase and ATPase activity. ATP is the best substrate, and Mg2+ is the most efficient divalent metal ion for helicase activity. A Lys to His mutation in the purine nucleotide-binding site results in a protein that inhibits helicase activity in a dominant negative manner. Rep40 unwinds double-stranded DNA containing a 3' single-stranded end, or blunt end, unlike the Rep68 and Rep52 enzymes, which have a strict requirement for DNA duplexes containing a 3' single-stranded end. Values for KATP in the ATPase assay are 1.1 +/- 0.2 mM and 1.2 +/- 0.2 mM in the absence and presence, respectively, of single-stranded DNA. Values for Vmax are 220 +/- 10 and 1,500 +/- 90 nmol/min/mg in the absence and presence, respectively, of single-stranded DNA. These studies provide the first enzymatic characterization of the AAV Rep40 protein and elucidate important functional differences between the AAV helicases.     

DNA-induced dimerization of the Escherichia coli Rep helicase.

The Escherichia coli Rep protein is a DNA helicase that is involved in DNA replication. We have examined the effects of DNA binding on the assembly state of the Rep protein using small-zone gel permeation chromatography and chemical crosslinking of the protein. Complexes of Rep protein were formed with short single-stranded and duplex hairpin oligodeoxynucleotides with lengths such that only a single Rep monomer could bind per oligodeoxynucleotide (i.e. 2 Rep monomers could not bind contiguously on the oligodeoxynucleotides). In the absence of DNA, Rep protein is monomeric (Mr 72,800) up to concentrations of at least 8 microM (monomer), even in the presence of its nucleotide cofactors (ATP, ADP, ATP-gamma-S). However, the binding of Rep monomers to single-stranded (ss) oligodeoxynucleotides, d(pN)n (12 less than or equal to n less than or equal to 20), induces the Rep monomers to oligomerize. Upon treatment of the Rep-ss oligodeoxynucleotide complexes with the protein crosslinking reagent dimethyl-suberimidate (DMS) and subsequent removal of the DNA, crosslinked Rep dimers are observed, independent of oligodeoxynucleotide length (n less than or equal to 20). Furthermore, short duplex oligodeoxynucleotides also induce the Rep monomers to dimerize. Formation of the Rep dimers results from an actual DNA-induced dimerization, rather than the adventitious crosslinking of Rep monomers bound contiguously to a single oligodeoxynucleotide. The purified DMS-crosslinked Rep dimer shows increased affinity for DNA and retains DNA-dependent ATPase and DNA helicase activities, as shown by its ability to unwind M13 RF DNA in the presence of the bacteriophage f1 gene II protein. On the basis of these observations and since the dimer is the major species when Rep is bound to DNA, we suggest that a DNA-induced Rep dimer is the functionally active form of the Rep helicase.     

Escherichia coli DNA helicase I catalyzes a unidirectional and highly processive unwinding reaction

Helicase I has been purified to greater than 95% homogeneity from an F+ strain of Escherichia coli, and characterized as a single-stranded DNA-dependent ATPase and a helicase. The duplex DNA unwinding reaction requires a region of ssDNA for enzyme binding and concomitant nucleoside 5'-triphosphate hydrolysis. All eight predominant nucleoside 5'-triphosphates can satisfy this requirement. Unwinding is unidirectional in the 5' to 3' direction. The length of duplex DNA unwound is independent of protein concentration suggesting that the unwinding reaction is highly processive. Kinetic analysis of the unwinding reaction indicates that the enzyme turns over very slowly from one DNA substrate molecule to another. The ATP hydrolysis reaction is continuous when circular partial duplex DNA substrates are used as DNA effectors. When linear partial duplex substrates are used ATP hydrolysis is barely detectable, although the kinetics of the unwinding reaction on linear partial duplex substrates are identical to those observed using a circular partial duplex DNA substrate. This suggests that ATP hydrolysis fuels continuous translocation of helicase I on circular single-stranded DNA while on linear single stranded DNA the enzyme translocates to the end of the DNA molecule where it must slowly dissociate from the substrate molecule and/or slowly associate with a new substrate molecule, thus resulting in a very low rate of ATP hydrolysis.