Simian-virus-40 large-T-antigen-catalyzed DNA and RNA unwinding reactions

Simian virus 40 large T antigen is a helicase separating the complementary strands of double-stranded DNA in the presence of hydrolyzable ATP and of double-stranded RNA in the presence of non-ATP nucleotides (GTP, CTP or UTP). We have constructed partially single-stranded nucleic acid substrates consisting of RNA or DNA strands hydrogen bonded to either RNA or DNA complements. We found that ATP is utilized as a cofactor for the T-antigen-catalyzed unwinding reaction when the substrates contain overhanging single-stranded DNA, regardless of whether the double-stranded region is DNA or hybrid DNA.RNA. Conversely, non-ATP nucleotides are used when the overhanging single strand is RNA. Based on these and additional findings, we propose that the bound nucleic acid induces a conformational change in T antigen resulting in a proper orientation of both nucleic acid and nucleotide relative to the active center of the ATPase/helicase domain of the enzyme. The implications of our conclusion for the roles which T antigen may play in vivo are discussed.     

Simian virus 40 large T antigen DNA helicase. Characterization of the ATPase-dependent DNA unwinding activity and its substrate requirements

The ATPase of SV40 large T antigen (T antigen) which is essential for the replication of SV40 minichromosomes was recently shown to be functionally related to a newly discovered DNA helicase activity. The T antigen helicase unwinds DNA duplices of several kilobase pairs in a reaction depending on the presence of hydrolyzable ribo- or deoxyribonucleoside triphosphates. The in vitro rate of movement through duplex DNA was found to be about 100 base pairs/min at 37 degrees C. For DNA unwinding, T antigen requires a 3'-single strand extension of a partially double-stranded substrate and invades the double strand section processively, in a 3' to 5' direction. The minimum length of the single-stranded tail was determined to be less than 5 nucleotides. Unwinding studies in the presence of the Escherichia coli single strand-specific DNA-binding protein and competition experiments indicate that T antigen helicase binds preferentially at the single-stranded/double-stranded DNA junction. This DNA structure is therefore proposed to serve as an entry site for the T antigen helicase. Previously reported data suggest that T antigen is the replicative helicase of the SV40 minichromosome. The results presented here are consistent with these findings and imply that T antigen migrates actively and processively along the template for the leading strand.     

The helicase activity associated with hepatitis C virus nonstructural protein 3 (NS3).

To assess the RNA helicase activity of hepatitis C virus (HCV) nonstructural protein 3 (NS3), a polypeptide encompassing amino acids 1175 to 1657, which cover only the putative helicase domain, was expressed in Escherichia coli by a pET expression vector. The protein was purified to near homogeneity and assayed for RNA helicase activity in vitro with double-stranded RNA substrates prepared from a multiple cloning sequence and an HCV 5' nontranslated region (5'-NTR) or 3'-NTR. The enzyme acted successfully on substrates containing both 5' and 3' single-stranded regions (standard) or on substrates containing only the 3' single-stranded regions (3'/3') but failed to act on substrates containing only the 5' single-stranded regions (5'/5') or on substrates lacking the single-stranded regions (blunt). These results thus suggest 3' to 5' directionality for HCV RNA helicase activity. However, a 5'/5' substrate derived from the HCV 5'-NTR was also partially unwound by the enzyme, possibly because of unique properties inherent in the 5' single-stranded regions. Gel mobility shift analyses demonstrated that the HCV NS3 helicase could bind to either 5'- or 3'-tailed substrates but not to substrates lacking a single-stranded region, indicating that the polarity of the RNA strand to which the helicase bound was a more important enzymatic activity determinant. In addition to double-stranded RNA substrates, HCV NS3 helicase activity could displace both RNA and DNA oligonucleotides on a DNA template, suggesting that HCV NS3 too was disposed to DNA helicase activity. This study also demonstrated that RNA helicase activity was dramatically inhibited by the single-stranded polynucleotides. Taken altogether, our results indicate that the HCV NS3 helicase is unique among the RNA helicases characterized so far.     

The unwinding of duplex regions in DNA by the simian virus 40 large tumor antigen-associated DNA helicase activity

The DNA helicase activity associated with purified simian virus 40 (SV40) large tumor (T) antigen has been examined. A variety of DNA substrates were used to characterize this ATP-dependent activity. Linear single-stranded M13 DNA containing short duplex regions at both ends was used to show that SV40 T antigen helicase displaced the short, annealed fragment by unwinding in a 3' to 5' direction. Three different partial duplex structures consisting of 71-, 343-, and 851-nucleotide long fragments annealed to M13 single-stranded circular DNA were used to show that SV40 T antigen can readily unwind short and long duplex regions with almost equal facility. ATP and MgCl2 were required for this reaction. With the exception of GTP, dGTP, and CTP, the other common nucleoside triphosphates substituted for ATP with varied efficiency, while adenosine 5'-O-(thiotriphosphate) was inactive. The T antigen helicase activity was also examined using completely duplex DNA fragments (approximately 300 base pairs) with or without the SV40 origin sequence as substrates. In reactions containing small amounts (0.6 ng) of DNA, the ATP-dependent unwinding of duplex DNA fragments occurred with no dependence on the origin sequence. This reaction was stimulated 5- to 6-fold by the addition of the Escherichia coli single-stranded DNA-binding protein. When competitor DNA was added so that the ratio of SV40 T antigen to DNA was reduced 1000-fold, only DNA fragments containing a functional SV40 origin of replication were unwound. This reaction was dependent on ATP, MgCl2, and a DNA-binding protein, and was stimulated by inorganic phosphate or creatine phosphate. The origin sequence requirements for the unwinding reaction were the same as those for replication (the 64-base pair sequence present at T antigen binding site 2). Thus, under specified conditions, only duplex DNA fragments containing an intact SV40 core origin were unwound. In contrast, unwinding of partially duplex segments of DNA flanked by single-stranded regions can occur with no sequence specificity.     

DNA helicase and nucleoside-5'-triphosphatase activities of polyoma virus large tumor antigen

Polyoma virus large tumor antigen (PyV T antigen) has been purified to near homogeneity by immunoaffinity column chromatography. We have detected DNA helicase and ATPase (nucleoside-5'-triphosphatase) activities in the purified PyV T antigen fraction and characterized these activities. The ATPase activity was stimulated about 2-fold by poly(dT), which was the most effective stimulator among the synthetic polynucleotides tested. Natural nucleic acids, such as calf thymus native and heat-denatured DNA, and single-stranded circular fd DNA were also effective, but the degree of stimulation was less than 1.5-fold. The basal and poly(dT)-stimulated ATPase activities showed similar preference for nucleoside 5'-triphosphates, requirement for divalent cations, and pH optima. The preference for nucleoside 5'-triphosphates was ATP, dATP greater than CTP, UTP much greater than GTP. The only difference observed between the two activities was salt sensitivity. The basal ATPase activity was resistant to KC1 up to 300 mM. In contrast, poly-(dT)-stimulated activity was reduced to the level of basal activity at 300 mM KC1. DNA helicase activity required divalent cations and was dependent on hydrolysis of ATP. The activity showed similar preference for nucleoside 5'-triphosphates, requirement for divalent cations, and pH optimum as the two ATPase activities, and the salt sensitivity of DNA helicase activity was similar to that of poly(dT)-stimulated ATPase activity. The helicase activity was inhibited competitively by the addition of single-stranded or double-stranded DNA, and a relatively high inhibitory activity was observed with poly [d(A-T)]. The PyV T antigen helicase was found to migrate in the 3' to 5' direction along the DNA strand to which the protein bound.     

p53-catalyzed annealing of complementary single-stranded nucleic acids.

p53 has been reported to inhibit the DNA helicase intrinsic to simian virus 40 large tumor antigen (T antigen). We found that inhibition is not restricted to T antigen, but also affects several other DNA and RNA helicases. Complexing of the helicases by the p53 protein as a possible inactivation mechanism could be excluded. Instead, the anti-helicase activity can be explained by our finding that p53 binds with high affinity to single-stranded nucleic acids and has a strong DNA.DNA and RNA.RNA annealing activity. We could also show that p53 is able to alter the secondary structure of RNA and/or to influence dynamic RNA-RNA interactions. These results, and the fact that the affinity of p53 to RNA is about one order of magnitude higher than to single-stranded DNA, imply an RNA-specific function of p53 in vivo.     

Sequence independent duplex DNA opening reaction catalysed by SV40 large tumor antigen.

Simian virus 40 (SV40) large tumor antigen (T antigen) is mainly localized in the nucleus where it exhibits two biochemical properties: DNA binding and helicase activity. Both activities are necessary for viral DNA replication and may also enable T antigen to modulate cellular growth. Here we present biochemical and electron microscopic evidence that the helicase activity can start at internal sites of fully double-stranded DNA molecules not containing the SV40 origin or replication. Using T antigen specific monoclonal antibodies, this unwinding reaction can be biochemically divided in an initiation (duplex opening) and a propagation step. The duplex opening reaction (as well as the propagation step) does not depend on a specific DNA sequence or secondary structure. In addition, we have found that T antigen forms an ATP dependent nucleoprotein complex at double-stranded DNA, which may be an essential step for the sequence independent duplex DNA opening reaction.     

Crystal structure of the hexameric replicative helicase RepA of plasmid RSF1010

Unwinding of double-stranded DNA into single-stranded intermediates required for various fundamental life processes is catalyzed by helicases, a family of mono-, di- or hexameric motor proteins fueled by nucleoside triphosphate hydrolysis. The three-dimensional crystal structure of the hexameric helicase RepA encoded by plasmid RSF1010 has been determined by X-ray diffraction at 2.4 A resolution. The hexamer shows an annular structure with 6-fold rotational symmetry and a approximately 17 A wide central hole, suggesting that single-stranded DNA may be threaded during unwinding. Homologs of all five conserved sequence motifs of the DnaB-like helicase family are found in RepA, and the topography of the monomer resembles RecA and the helicase domain of the bacteriophage T7 gp4 protein. In a modeled complex, ATP molecules are located at the subunit interfaces and clearly define adenine-binding and ATPase catalytic sites formed by amino acid residues located on adjacent monomers; most remarkable is the "arginine finger" Arg207 contributing to the active site in the adjacent monomer. This arrangement of active-site residues suggests cooperativity between monomers in ATP hydrolysis and helicase activity of RepA. The mechanism of DNA unwinding remains elusive, as RepA is 6-fold symmetric, contrasting the recently published asymmetric structure of the bacteriophage T7 gp4 helicase domain.     

Simian virus 40 (SV40) T antigen binds specifically to double-stranded DNA but not to single-stranded DNA or DNA/RNA hybrids containing the SV40 regulatory sequences.

Simian virus 40 T antigen has been shown previously to bind specifically with high affinity to sites within the regulatory region of double-stranded simian virus 40 DNA. Using competition filter binding and the DNA-binding immunoassay, we show that T antigen did not bind specifically to either early or late single-stranded DNA containing these binding sites. Moreover, T antigen did not bind these sequences present in single-stranded RNA, RNA/RNA duplexes, or RNA/DNA hybrids. T antigen did, however, bind as efficiently to single-stranded DNA-cellulose as to double-stranded DNA-cellulose. This binding was nonspecific because it was independent of the presence of T-antigen-binding sites. The implications of these observations are discussed.     

Purification and characterization of delta helicase from fetal calf thymus.

A DNA helicase (delta helicase) which partially copurifies with DNA polymerase delta has been highly purified from fetal calf thymus. delta helicase differs in physical and enzymatic properties from other eukaryotic DNA helicases described thus far. The enzyme has an apparent mass of 57 kDa by gel filtration and is associated with polypeptides of 56 and 52 kDa by SDS-polyacrylamide gel electrophoresis. Photo-cross-linking of the purified enzyme with [alpha-32P]ATP resulted in labeling of a polypeptide of approximately 58 kDa, suggesting that the active site is present on the larger polypeptide. Unwinding of a partial duplex requires a nucleoside triphosphate which can be either ATP or dATP but not a nonhydrolyzable analogue of ATP. Other ribo- and deoxyribonucleoside triphosphates have little or no activity as cofactors. delta helicase also has DNA-dependent ATPase activity which has a relatively low Km for ATP (40 microM). delta helicase binds to single-stranded DNA but has little or no affinity for double-stranded DNA or single-stranded RNA. Similar to replicative DNA helicases from prokaryotes and the herpes simplex virus type 1 helicase-primase, delta helicase translocates in the 5'-3' direction along the strand to which it is bound and preferentially unwinds DNA substrates with a forklike structure.     

Mapping of helicase and helicase substrate-binding domains on simian virus 40 large T antigen.

We generated fragments of simian virus 40 large tumor antigen (T antigen) by tryptic digestion and assayed them for helicase activity and helicase substrate (mostly single-stranded DNA)-binding activity in order to map the domain sites on the protein. The N-terminal 130 amino acids were not required for either activity, since a 76-kilodalton (kDa) fragment (amino acids 131 to 708) was just as active as intact T antigen. To map the helicase domain further, smaller tryptic fragments were generated. A 66-kDa fragment (131 to about 616) retained some activity, whereas a slightly smaller 62-kDa fragment (137 or 155 to 616) had none. This suggests that the minimal helicase domain maps from residue 131 to approximately residue 616. To map the helicase substrate-binding domain, we tested various fragments in a substrate-binding assay. The smallest fragment for which we could clearly demonstrate activity was a 46-kDa fragment (131 to 517). To determine the relationship between the helicase substrate domain and the origin-binding domain (131 to 257, minimal core region; 131 to 371, optimal region), we performed binding experiments with competitor DNAs present. We found that origin-containing double-stranded DNA was an excellent competitor of the binding of the helicase substrate to T antigen, suggesting that the two domains overlap. Therefore, full helicase activity requires at least a partial origin-binding domain as well as an active ATPase domain. Additionally, we found that the helicase substrate was a poor competitor of origin-binding activity, indicating that T antigen has a much higher affinity to origin sequences than to the helicase substrate.     

The MER3 helicase involved in meiotic crossing over is stimulated by single-stranded DNA-binding proteins and unwinds DNA in the 3' to 5' direction

The meiosis-specific MER3 protein of Saccharomyces cerevisiae is required for crossing over, which ensures faithful segregation of homologous chromosomes at the first meiotic division. The predicted sequence of the MER3 protein contains the seven motifs characteristic of the DExH-box type of DNA/RNA helicases. The purified MER3 protein is a DNA helicase, which can displace a 50-nucleotide fragment annealed to a single-stranded circular DNA. MER3 was found to have ATPase activity, which was stimulated either by single- or double-stranded DNA. The turnover rate, k(cat), of ATP hydrolysis was approximately 500/min in the presence of either DNA. MER3 was able to efficiently displace relatively long 631-nucleotide fragments from single-stranded circular DNA only in the presence of the S. cerevisiae single-stranded DNA-binding protein, RPA (replication protein A). It appears that RPA inhibits re-annealing of the single-stranded products of the MER3 helicase. The MER3 helicase was found to unwind DNA in the 3' to 5' direction relative to single-stranded regions in the DNA substrates. Possible roles for the MER3 helicase in meiotic crossing over are discussed.     

Staphylococcus aureus DinG, a helicase that has evolved into a nuclease

DinG (damage inducible gene G) is a bacterial superfamily 2 helicase with 5′→3′ polarity. DinG is related to the XPD (xeroderma pigmentosum complementation group D) helicase family, and they have in common an FeS (iron–sulfur)-binding domain that is essential for the helicase activity. In the bacilli and clostridia, the DinG helicase has become fused with an N-terminal domain that is predicted to be an exonuclease. In the present paper we show that the DinG protein from Staphylococcus aureus lacks an FeS domain and is not a DNA helicase, although it retains DNA-dependent ATP hydrolysis activity. Instead, the enzyme is an active 3′→5′ exonuclease acting on single-stranded DNA and RNA substrates. The nuclease activity can be modulated by mutation of the ATP-binding cleft of the helicase domain, and is inhibited by ATP or ADP, suggesting a modified role for the inactive helicase domain in the control of the nuclease activity. By degrading rather than displacing RNA or DNA strands, the S. aureus DinG nuclease may accomplish the same function as the canonical DinG helicase.     

Nonspecific double-stranded DNA binding activity of simian virus 40 large T antigen is involved in melting and unwinding of the origin

Helicase activity is required for T antigen to unwind the simian virus 40 origin. We previously mapped this activity to residues 131 and 616. In this study, we generated a series of mutants with single-point substitutions in the helicase domain to discover other potential activities required for helicase function. A number of DNA unwinding-defective mutants were generated. Four of these mutants (456RA, 460ED, 462GA, and 499DA) were normal in their ability to hydrolyze ATP and were capable of associating into double hexamers in the presence of origin DNA. Furthermore, they possessed normal ability to bind to single-stranded DNA. However, they were severely impaired in unwinding origin-containing DNA fragments and in carrying out a helicase reaction with an M13 partial duplex DNA substrate. Interestingly, these mutants retained some ability to perform a helicase reaction with artificial replication forks, indicating that their intrinsic helicase activity was functional. Intriguingly, these mutants had almost completely lost their ability to bind to double-stranded DNA nonspecifically. The mutants also failed to melt the early palindrome region of the origin. Taken together, these results indicate that the mutations have destroyed a novel activity required for unwinding of the origin. This activity depends on the ability to bind to DNA nonspecifically, and in its absence, T antigen is unable to structurally distort and subsequently unwind the origin.     

Simian virus 40 large T antigen untwists DNA at the origin of DNA replication.

Simian virus 40 large tumor antigen (SV40 T antigen) untwists DNA at the SV40 replication origin. In the presence of ATP, T antigen shifted the average linking number of an SV40 origin-containing plasmid topoisomer distribution. The loss of up to two helical turns was detected. The reaction required the presence of the 64-base pair core origin of replication containing T antigen DNA binding site II; binding site I had no effect on the untwisting reaction. The presence of human single-stranded DNA binding protein (SSB) slightly reduced the degree of untwisting in the presence of ATP. ATP hydrolysis was not required since untwisting occurred in the presence of nonhydrolyzable analogs of ATP. However, in the presence of a nonhydrolyzable analog of ATP, the requirement for the SV40 origin sequence was lost. The origin requirement for DNA untwisting was also lost in the absence of dithiothreitol. The origin-specific untwisting activity of T antigen is distinct from its DNA helicase activity, since helicase activity does not require the SV40 origin but does require ATP hydrolysis. The lack of a requirement for SSB or ATP hydrolysis and the reduction in the pitch of the DNA helix by just a few turns at the replication origin distinguishes this reaction from the T antigen-mediated DNA unwinding reaction, which results in the formation of a highly underwound DNA molecule. Untwisting occurred without a lag after the start of the reaction, whereas unwound DNA was first detected after a lag of 10 min. It is proposed that the formation of a multimeric T antigen complex containing untwisted DNA at the SV40 origin is a prerequisite for the initiation of DNA unwinding and replication.     

Amplification of snap-back DNA synthesis reactions by the uvsX recombinase of bacteriophage T4.

The uvsX protein of bacteriophage T4 is a recA-type recombinase. This protein has previously been shown to help initiate DNA replication on a double-stranded DNA template by catalyzing synapsis between the template and a homologous DNA single strand that serves as primer. Here, we demonstrate that this replication-initiating activity of the uvsX protein greatly amplifies the snap-back (hairpin-primed) DNA synthesis that is catalyzed by the T4 DNA polymerase holoenzyme on linear, single-stranded DNA templates. Amplification requires the presence of uvsX protein, the DNA polymerase holoenzyme, T4 gene 32 protein, and a T4 DNA helicase, in a reaction that is modulated by the T4 uvsY protein (an accessory protein to the uvsX recombinase). The reaction products consist primarily of large networks of double-stranded and single-stranded DNA. With alkali or heat treatment, these networks resolve into dimer-length single-stranded DNA chains that renature instantaneously to reform a monomer-length double helix. A simple model can explain this uvsX protein-dependent amplification of snap-back DNA synthesis; the mechanism proposed makes several predictions that are confirmed by our experiments.     

The ATPase, RNA unwinding, and RNA binding activities of recombinant p68 RNA helicase

p68 RNA helicase, a nuclear RNA helicase, was identified 2 decades ago. The protein plays very important roles in cell development and organ maturation. However, the biological functions and enzymology of p68 RNA helicase are not well characterized. We report the expression and purification of recombinant p68 RNA helicase in a bacterial system. The recombinant p68 is an ATP-dependent RNA helicase. ATPase assays demonstrated that double-stranded RNA (dsRNA) is much more effective than single-stranded RNA in stimulating ATP hydrolysis by the recombinant protein. Consistently, RNA-binding assays showed that p68 RNA helicase binds single-stranded RNA weakly in an ATP-dependent manner. On the other hand, the recombinant protein has very high affinity for dsRNA. Binding of the protein to dsRNA is ATP-independent. The data indicate that p68 may directly target dsRNA as its natural substrate. Interestingly, the recombinant p68 RNA helicase unwinds dsRNA in both 3' --> 5' and 5' --> 3' directions. This is the second example of a Asp-Glu-Ala-Asp (DEAD) box RNA helicase that unwinds RNA duplexes in a bi-directional manner.     

Electron microscopic observation and single-stranded DNA binding activity of the Mcm4,6,7 complex

Mcm2-7 proteins that play an essential role in eukaryotic DNA replication contain DNA-dependent ATPase motifs in a central domain that, from yeast to mammals, is highly conserved. Our group has reported that a DNA helicase activity is associated with a 600 kDa human Mcm4, 6 and 7 complex. The structure of the Mcm4,6,7 complex was visualized by electron microscopy after negative staining with uranyl acetate. The complex contained toroidal forms with a central channel and also contained structures with a slit. Gel-shift analysis indicated that the level of affinity of the Mcm4,6,7 complex for single-stranded DNA was comparable to that of SV40 T antigen, although the Mcm4,6,7 complex required longer single-stranded DNA for the binding than did SV40 T antigen. The nucleoprotein complexes of Mcm4,6,7 and single-stranded DNA were visualized as beads in a queue or beads on string-like structures. The formation of these nucleoprotein complexes was erased by Mcm2 that is a potential inhibitor of the Mcm4,6,7 helicase. We also found that the DNA helicase activity of Mcm4,6,7 complex was inhibited by the binding of Mcm3,5 complex. These results support the notion that the Mcm4,6,7 complex functions as a DNA helicase and the formation of 600 kDa complex is essential for the activity.     

Vaccinia virus RNA helicase: nucleic acid specificity in duplex unwinding.

Vaccinia virus RNA helicase (NPH-II) catalyzes nucleoside triphosphate-dependent unwinding of duplex RNAs containing a single-stranded 3' RNA tail. In this study, we examine the structural features of the nucleic acid substrate that are important for helicase activity. Strand displacement was affected by the length of the 3' tail. Whereas NPH-II efficiently unwound double-stranded RNA substrates with 19- or 11-nucleotide (nt) 3' tails, shortening the 3' tail to 4 nt reduced unwinding by an order of magnitude. Processivity of the helicase was inferred from its ability to unwind a tailed RNA substrate containing a 96-bp duplex region. NPH-II exhibited profound asymmetry in displacing hybrid duplexes composed of DNA and RNA strands. A 34-bp RNA-DNA hybrid with a 19-nt 3' RNA tail was unwound catalytically, whereas a 34-bp DNA-RNA hybrid containing a 19-nt 3' DNA tail was 2 orders of magnitude less effective as a helicase substrate. NPH-II was incapable of displacing a 34-bp double-stranded DNA substrate of identical sequence. 3'-Tailed DNA molecules with 24- or 19-bp duplex regions were also inert as helicase substrates. On the basis of current models for RNA-DNA hybrid structures, we suggest the following explanation for these findings. (i) Unwinding of duplex nucleic acids by NPH-II is optimal when the polynucleotide strand of the duplex along which the enzyme translocates has adopted an A-form secondary structure, and (ii) a B-form secondary structure impedes protein translocation through DNA duplexes.