Partial Duplex Substrates Research Articles

Biochemical analysis of SARS-CoV-2 Nsp13 helicase implicated in COVID-19 and factors that regulate its catalytic functions

Replication of the 30 kilobase genome of SARS-CoV-2, responsible for COVID-19, is a key step in the coronavirus life cycle that requires a set of virally encoded nonstructural proteins such as the highly conserved Nsp13 helicase. However, the features that contribute to catalytic properties of Nsp13 are not well established. Here, we biochemically characterized the purified recombinant SARS-CoV-2 Nsp13 helicase protein, focusing on its catalytic functions, nucleic acid substrate specificity, nucleotide/metal cofactor requirements, and displacement of proteins from RNA molecules proposed to be important for its proofreading role during coronavirus replication. We determined that Nsp13 preferentially interacts with single-stranded DNA compared to single-stranded RNA to unwind a partial duplex helicase substrate. We present evidence for functional cooperativity as a function of Nsp13 concentration, which suggests that oligomerization is important for optimal activity. In addition, under single-turnover conditions, Nsp13 unwound partial duplex RNA substrates of increasing double-stranded regions (16 - 30 base pairs) with similar efficiency, suggesting the enzyme unwinds processively in this range. We also show Nsp13-catalyzed RNA unwinding is abolished by a site-specific neutralizing linkage in the sugar-phosphate backbone, demonstrating continuity in the helicase-translocating strand is essential for unwinding the partial duplex substrate. Taken together, we demonstrate for the first time that coronavirus helicase Nsp13 disrupts a high affinity RNA-protein interaction in a uni-directional and ATP-dependent manner. Furthermore, sensitivity of Nsp13 catalytic functions to Mg2+ concentration suggests a regulatory mechanism for ATP hydrolysis, duplex unwinding, and RNA protein remodeling, processes implicated in SARS-CoV-2 replication and proofreading.

Yeast Pif1 accelerates annealing of complementary DNA strands.

Pif1 is a helicase involved in the maintenance of nuclear and mitochondrial genomes in eukaryotes. Here we report a new activity of Saccharomyces cerevisiae Pif1, annealing of complementary DNA strands. We identified preferred substrates for annealing as those that generate a duplex product with a single-stranded overhang relative to a blunt end duplex. Importantly, we show that Pif1 can anneal DNA in the presence of ATP and Mg2+. Pif1-mediated annealing also occurs in the presence of single-stranded DNA binding proteins. Additionally, we show that partial duplex substrates with 3′-single-stranded overhangs such as those generated during double-strand break repair can be annealed by Pif1.

Abstract 5361: Development of targeted inhibitors against RecQ1 helicase

Abstract Background: RecQ helicases are a ubiquitous family of DNA unwinding enzymes involved in the maintenance of chromosome stability. RecQ1 helicase genomic variations have an important prognostic role and represents a therapeutic target in pancreatic cancer [JCO 2006; 24 (11) 1720-1728]. Pubchem data indicated potential RecQ1 activity >600 compounds using a qHTS RecQ1 assay (NIH Chemical Genomics Center). Methods: We applied a set of filters including molecular weight, potency and Hill curve fitting and also removed those interfering with biological assay to identify sixteen compounds for studying RecQ1 inhibition. Two compounds showed strong inhibition of RecQ1 helicase activity at 50 µM and at IC50 < 5 µM, respectively. From a large library of compounds, eight potential inhibitors were synthesized in three simple steps. First, sulfonylation of various amines with 3-Nitrobenzenesulfonyl chloride was performed followed by reduction of the nitro group into an amine. Finally, acylation with variety of acid chlorides resulted in the final library of RecQ1 analogs. These agents were studied for RecQ1 helicase activity assay with the radiolabeled partial duplex substrate in vitro. The RecQ1 inhibitor candidate compounds were investigated in pancreatic cancer cell lines (AsPC-1, BxPC-3, Capan-1, PANC-1, and Mia-Paca2) for anti-tumor efficacy using cell proliferation assay with CellTiter-Glo® luminescence. Combination studies with test compounds and poly (ADP ribose) polymerase (PARP) inhibitor olaparib and topoisomerase Inhibitor SN38 were performed. Results: Though none of the compounds showed the same level of activity of the parent compounds, we were able to demonstrate a modest level of helicase activity with several. Two compounds displayed strong inhibition of pancreatic cancer cell proliferation. Two other compounds displayed synergistic inhibition with SN38 or olaparib of pancreatic cancer cell proliferation and induced DNA damage. Conclusions: Synthesis of RecQ1 inhibitors is feasible and shows potential for anti-tumor efficacy. Note: This abstract was not presented at the meeting. Citation Format: Mingxin Zuo, David Maxwell, Basvoju A. Bhanu Prasad, Zhenghong Peng, William Bornmann, Milind M. Javle. Development of targeted inhibitors against RecQ1 helicase. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5361. doi:10.1158/1538-7445.AM2014-5361

The telomere resolvase of the Lyme disease spirochete, Borrelia burgdorferi, promotes DNA single-strand annealing and strand exchange

Spirochetes of the genus Borrelia include the tick-transmitted causative agents of Lyme disease and relapsing fever. They possess unusual genomes composed mainly of linear replicons terminated by closed DNA hairpin telomeres. Hairpin telomeres present an uninterrupted DNA chain to the replication machinery overcoming the ‘end-replication problem’ for the linear replicons. Hairpin telomeres are formed from inverted repeat replicated telomere junctions by the telomere resolvase, ResT. ResT uses a reaction mechanism similar to that of the type IB topoisomerases and tyrosine recombinases. We report here that ResT also possesses single-strand annealing activity and a limited ability to promote DNA strand exchange reactions on partial duplex substrates. This combination of activities suggests ResT is a nexus between the seemingly distinct processes of telomere resolution and homologous recombination. Implications for hairpin telomere replication and linear plasmid recombination, including antigenic variation, are discussed.

Molecular Basis for Recognition of Nucleoside Triphosphate by Gene 4 Helicase of Bacteriophage T7

The translocation of DNA helicases on single-stranded DNA and the unwinding of double-stranded DNA are fueled by the hydrolysis of nucleoside triphosphates (NTP). Although most helicases use ATP in these processes, the DNA helicase encoded by gene 4 of bacteriophage T7 uses dTTP most efficiently. To identify the structural requirements of the NTP, we determined the efficiency of DNA unwinding by T7 helicase using a variety of NTPs and their analogs. The 5-methyl group of thymine was critical for the efficient unwinding of DNA, although the presence of a 3'-ribosyl hydroxyl group partially overcame this requirement. The NTP-binding pocket of the protein was examined by randomly substituting amino acids for several amino acid residues (Thr-320, Arg-504, Tyr-535, and Leu-542) that the crystal structure suggests interact with the nucleotide. Although positions 320 and 542 required aliphatic residues of the appropriate size, an aromatic side chain was necessary at position 535 to stabilize NTP for efficient unwinding. A basic side chain of residue 504 was essential to interact with the 4-carbonyl of the thymine base of dTTP. Replacement of this residue with a small aliphatic residue allowed the accommodation of other NTPs, resulting in the preferential use of dATP and the use of dCTP, a nucleotide not normally used. Results from this study suggest that the NTP must be stabilized by specific interactions within the NTP-binding site of the protein to achieve efficient hydrolysis. These interactions dictate NTP specificity.

Modulation of UvrD Helicase Activity by Covalent DNA-Protein Cross-links

UvrD (DNA helicase II) has been implicated in DNA replication, DNA recombination, nucleotide excision repair, and methyl-directed mismatch repair. The enzymatic function of UvrD is to translocate along a DNA strand in a 3' to 5' direction and unwind duplex DNA utilizing a DNA-dependent ATPase activity. In addition, UvrD interacts with many other proteins involved in the above processes and is hypothesized to facilitate protein turnover, thus promoting further DNA processing. Although UvrD interactions with proteins bound to DNA have significant biological implications, the effects of covalent DNA-protein cross-links on UvrD helicase activity have not been characterized. Herein, we demonstrate that UvrD-catalyzed strand separation was inhibited on a DNA strand to which a 16-kDa protein was covalently bound. Our sequestration studies suggest that the inhibition of UvrD activity is most likely due to a translocation block and not helicase sequestration on the cross-link-containing DNA substrate. In contrast, no inhibition of UvrD-catalyzed strand separation was apparent when the protein was linked to the complementary strand. The latter result is surprising given the earlier observations that the DNA in this covalent complex is severely bent ( approximately 70 degrees ), with both DNA strands making multiple contacts with the cross-linked protein. In addition, UvrD was shown to be required for replication of plasmid DNAs containing covalent DNA-protein complexes. Combined, these data suggest a critical role for UvrD in the processing of DNA-protein cross-links.

An in Trans Interaction at the Interface of the Helicase and Primase Domains of the Hexameric Gene 4 Protein of Bacteriophage T7 Modulates Their Activities

DNA helicase and primase are essential for DNA replication. The helicase unwinds the DNA to provide single-stranded templates for DNA polymerase. The primase catalyzes the synthesis of oligoribonucleotides for the initiation of lagging strand synthesis. The two activities reside in a single polypeptide encoded by gene 4 of bacteriophage T7. Their coexistence within the same polypeptide facilitates their coordination during DNA replication. One surface of helix E within the helicase domain is positioned to interact with the primase domain and the linker connecting the two domains within the functional hexamer. The interaction occurs in trans such that helix E interacts with the primase domain and the linker of the adjacent subunit. Most alterations of residues on the surface of helix E (Arg(404), Lys(408), Tyr(411), and Gly(415)) eliminate the ability of the altered proteins to complement growth of T7 phage lacking gene 4. Both Tyr(411) and Gly(415) are important in oligomerization of the protein. Alterations G415V and K408A simultaneously influence helicase and primase activities in opposite manners that mimic events observed during coordinated DNA synthesis. The results suggest that Asp(263) located in the linker of one subunit can interact with Tyr(411), Lys(408), or Arg(404) in helix E of the adjacent subunit depending on the oligomerization state. Thus the switch in contacts between Asp(263) and its three interacting residues in helix E of the adjacent subunit results in conformational changes that modulate helicase and primase activity.

Regulation of WRN Protein Cellular Localization and Enzymatic Activities by SIRT1-mediated Deacetylation

Werner syndrome is an autosomal recessive disorder associated with premature aging and cancer predisposition caused by mutations of the WRN gene. WRN is a member of the RecQ DNA helicase family with functions in maintaining genome stability. Sir2, an NAD-dependent histone deacetylase, has been proven to extend life span in yeast and Caenorhabditis elegans. Mammalian Sir2 (SIRT1) has also been found to regulate premature cellular senescence induced by the tumor suppressors PML and p53. SIRT1 plays an important role in cell survival promoted by calorie restriction. Here we show that SIRT1 interacts with WRN both in vitro and in vivo; this interaction is enhanced after DNA damage. WRN can be acetylated by acetyltransferase CBP/p300, and SIRT1 can deacetylate WRN both in vitro and in vivo. WRN acetylation decreases its helicase and exonuclease activities, and SIRT1 can reverse this effect. WRN acetylation alters its nuclear distribution. Down-regulation of SIRT1 reduces WRN translocation from nucleoplasm to nucleoli after DNA damage. These results suggest that SIRT1 regulates WRN-mediated cellular responses to DNA damage through deacetylation of WRN.

Srs2 Helicase of Saccharomyces cerevisiae Selectively Unwinds Triplet Repeat DNA

Trinucleotide repeat expansions are the mutational cause of at least 15 genetic diseases. In vitro, single-stranded triplet repeat DNA forms highly stable hairpins, depending on repeat sequence, and a strong correlation exists between hairpin-forming ability and the risk of expansion in vivo. Hairpins are viewed, therefore, as likely mutagenic precursors to expansions. If a helicase unwinds the hairpin, it would be less likely to expand. Previous work indicated that yeast Srs2 DNA helicase selectively blocks expansions in vivo (Bhattacharyya, S., and Lahue, R. S. (2004) Mol. Cell. Biol. 24, 7324-7330). For example, srs2 mutants, including an ATPase-defective point mutant, exhibit substantially higher expansion rates than wild type controls. In contrast, mutation of another helicase gene, SGS1, had little effect on expansion rates. These findings prompted the idea that Srs2 might selectively unwind triplet repeat hairpins. In this study, DNA helicase assays were performed with purified Srs2, Sgs1, and Escherichia coli UvrD (DNA helicase II). Srs2 shows substantially faster unwinding than Sgs1 or UvrD on partial duplex substrates containing (CTG) x (CTG) sequences, provided that Srs2 encounters the triplet repeat DNA immediately on entering the duplex. Srs2 was also faster at unwinding (CAG) x (CAG)- and (CCG) x (CCG)-containing substrates and an intramolecular (CTG) x (CTG) hairpin. In contrast, all three enzymes unwind about equally well control substrates with either Watson-Crick base pairs or mismatched substrates with non-CNG repeats. Overall, the selective unwinding activity of Srs2 on triplet repeat hairpin DNA helps explain the genetic evidence that Srs2, not the RecQ homolog Sgs1, is a preferred helicase for preventing expansions.

Physical and Functional Mapping of the Replication Protein A Interaction Domain of the Werner and Bloom Syndrome Helicases

The single-stranded DNA-binding protein replication protein A (RPA) interacts with several human RecQ DNA helicases that have important roles in maintaining genomic stability; however, the mechanism for RPA stimulation of DNA unwinding is not well understood. To map regions of Werner syndrome helicase (WRN) that interact with RPA, yeast two-hybrid studies, WRN affinity pull-down experiments and enzyme-linked immunosorbent assays with purified recombinant WRN protein fragments were performed. The results indicated that WRN has two RPA binding sites, a high affinity N-terminal site, and a lower affinity C-terminal site. Based on results from mapping studies, we sought to determine if the WRN N-terminal region harboring the high affinity RPA interaction site was important for RPA stimulation of WRN helicase activity. To accomplish this, we tested a catalytically active WRN helicase domain fragment (WRN(H-R)) that lacked the N-terminal RPA interaction site for its ability to unwind long DNA duplex substrates, which the wild-type enzyme can efficiently unwind only in the presence of RPA. WRN(H-R) helicase activity was significantly reduced on RPA-dependent partial duplex substrates compared with full-length WRN despite the presence of RPA. These results clearly demonstrate that, although WRN(H-R) had comparable helicase activity to full-length WRN on short duplex substrates, its ability to unwind RPA-dependent WRN helicase substrates was significantly impaired. Similarly, a Bloom syndrome helicase (BLM) domain fragment, BLM(642-1290), that lacked its N-terminal RPA interaction site also unwound short DNA duplex substrates similar to wild-type BLM, but was severely compromised in its ability to unwind long DNA substrates that full-length BLM helicase could unwind in the presence of RPA. These results suggest that the physical interaction between RPA and WRN or BLM helicases plays an important role in the mechanism for RPA stimulation of helicase-catalyzed DNA unwinding.

Analysis of the DNA Substrate Specificity of the Human BACH1 HelicaseAssociated with BreastCancer

We have investigated the DNA substrate specificity of BACH1 (BRCA1-associated C-terminal helicase). The importance of various DNA structural elements for efficient unwinding by purified recombinant BACH1 helicase was examined. The results indicated that BACH1 preferentially binds and unwinds a forked duplex substrate compared with a duplex flanked by only one single-stranded DNA (ssDNA) tail. In support of its DNA substrate preference, helicase sequestration studies revealed that BACH1 can be preferentially trapped by forked duplex molecules. BACH1 helicase requires a minimal 5 ' ssDNA tail of 15 nucleotides for unwinding of conventional duplex DNA substrates; however, the enzyme is able to catalytically release the third strand of the homologous recombination intermediate D-loop structure irrespective of DNA tail status. In contrast, BACH1 completely fails to unwind a synthetic Holliday junction structure. Moreover, BACH1 requires nucleic acid continuity in the 5 ' ssDNA tail of the forked duplex substrate within six nucleotides of the ssDNA-dsDNA junction to initiate efficiently DNA unwinding. These studies provide the first detailed information on the DNA substrate specificity of BACH1 helicase and provide insight to the types of DNA structures the enzyme is likely to act upon to perform its functions in DNA repair or recombination.

The HRDC domain of BLM is required for the dissolution of double Holliday junctions

Bloom's syndrome is a hereditary cancer-predisposition disorder resulting from mutations in the BLM gene. In humans, BLM encodes one of five members of the RecQ helicase family. One function of BLM is to act in concert with topoisomerase IIIalpha (TOPO IIIalpha) to resolve recombination intermediates containing double Holliday junctions by a process called double Holliday junction dissolution, herein termed dissolution. Here, we show that dissolution is highly specific for BLM among human RecQ helicases and critically depends upon a functional HRDC domain in BLM. We show that the HRDC domain confers DNA structure specificity, and is required for the efficient binding to and unwinding of double Holliday junctions, but not for the unwinding of a simple partial duplex substrate. Furthermore, we show that lysine-1270 of BLM, which resides in the HRDC domain and is predicted to play a role in mediating interactions with DNA, is required for efficient dissolution.

Maturation of Bacteriophage T4 Lagging Strand Fragments Depends on Interaction of T4 RNase H with T4 32 Protein Rather than the T4 Gene 45 Clamp

In the bacteriophage T4 DNA replication system, T4 RNase H removes the RNA primers and some adjacent DNA before the lagging strand fragments are ligated. This 5'-nuclease has strong structural and functional similarity to the FEN1 nuclease family. We have shown previously that T4 32 protein binds DNA behind the nuclease and increases its processivity. Here we show that T4 RNase H with a C-terminal deletion (residues 278-305) retains its exonuclease activity but is no longer affected by 32 protein. T4 gene 45 replication clamp stimulates T4 RNase H on nicked or gapped substrates, where it can be loaded behind the nuclease, but does not increase its processivity. An N-terminal deletion (residues 2-10) of a conserved clamp interaction motif eliminates stimulation by the clamp. In the crystal structure of T4 RNase H, the binding sites for the clamp at the N terminus and for 32 protein at the C terminus are located close together, away from the catalytic site of the enzyme. By using mutant T4 RNase H with deletions in the binding site for either the clamp or 32 protein, we show that it is the interaction of T4 RNase H with 32 protein, rather than the clamp, that most affects the maturation of lagging strand fragments in the T4 replication system in vitro and T4 phage production in vivo.

P53 Modulates RPA-Dependent and RPA-Independent WRN Helicase Activity

Werner syndrome is a hereditary disorder characterized by the early onset of age-related symptoms, including cancer. The absence of a p53-WRN helicase interaction may disrupt the signal to direct S-phase cells into apoptosis for programmed cell death and contribute to the pronounced genomic instability and cancer predisposition in Werner syndrome cells. Results from coimmunoprecipitation studies indicate that WRN is associated with replication protein A (RPA) and p53 in vivo before and after treatment with the replication inhibitor hydroxyurea or gamma-irradiation that introduces DNA strand breaks. Analysis of the protein interactions among purified recombinant WRN, RPA, and p53 proteins indicate that all three protein pairs bind with similar affinity in the low nanomolar range. In vitro studies show that p53 inhibits RPA-stimulated WRN helicase activity on an 849-bp M13 partial duplex substrate. p53 also inhibited WRN unwinding of a short (19-bp) forked duplex substrate in the absence of RPA. WRN unwinding of the forked duplex substrate was specific, because helicase inhibition mediated by p53 was retained in the presence of excess competitor DNA and was significantly reduced or absent in helicase reactions catalyzed by a WRN helicase domain fragment lacking the p53 binding site or the human RECQ1 DNA helicase, respectively. p53 effectively inhibited WRN helicase activity on model DNA substrate intermediates of replication/repair, a 5' ssDNA flap structure and a synthetic replication fork. Regulation of WRN helicase activity by p53 is likely to play an important role in genomic integrity surveillance, a vital function in the prevention of tumor progression.

A novel nuclear DNA helicase with high specific activity from Pisum sativum catalytically translocates in the 3'-->5' direction.

A novel ATP-dependent nuclear DNA unwinding enzyme from pea has been purified to apparent homogeneity and characterized. This enzyme is present at extremely low abundance and has the highest specific activity among plant helicases. It is a heterodimer of 54 and 66 kDa polypeptides as determined by SDS/PAGE. On gel filtration chromatography and glycerol gradient centrifugation it gives a native molecular mass of 120 kDa and is named as pea DNA helicase 120 (PDH120). The enzyme can unwind 17-bp partial duplex substrates with equal efficiency whether or not they contain a fork. It translocates unidirectionally along the bound strand in the 3'-->5' direction. The enzyme also exhibits intrinsic single-stranded DNA- and Mg2+-dependent ATPase activity. ATP is the most favoured cofactor but other NTPs and dNTPs can also support the helicase activity with lower efficiency (ATP > GTP = dCTP > UTP > dTTP > CTP > dATP > dGTP) for which divalent cation (Mg2+ > Mn2+) is required. The DNA intercalating agents actinomycin C1, ethidium bromide, daunorubicin and nogalamycin inhibit the DNA unwinding activity of PDH120 with Ki values of 5.6, 5.2, 4.0 and 0.71 micro Ms, respectively. This inhibition might be due to the intercalation of the inhibitors into duplex DNA, which results in the formation of DNA-inhibitor complexes that impede the translocation of PDH120. Isolation of this new DNA helicase should make an important contribution to our better understanding of DNA transaction in plants.

Biochemical characterization of the small isoform of Drosophila melanogaster RECQ5 helicase.

Recently the gene encoding a member of the RecQ helicase family, RecQ5, was cloned from the fruit fly, Drosophila melanogaster [J.J.Sekelsky, M.H.Brodsky, G.M. Rubin and R.S. Hawley (1999) Nucleic Acids Res., 27, 3762-3769]. The Drosophila RecQ5 transcript is alternatively spliced, like its human counterpart, to yield three protein isoforms. Two of these isoforms are almost identical and have a predicted molecular weight of 54 kDa. The third isoform is larger and contains, in addition to the helicase domain shared by all three isoforms, a long highly charged C-terminal region. A small isoform of the Drosophila RecQ5 protein (RECQ5) has been expressed in Escherichia coli and purified. The purified protein is a single-stranded DNA-stimulated ATPase (dATPase) and a 3'-->5' DNA helicase. Hydrolysis of the nucleotide cofactor is required for unwinding activity and dATP supported the unwinding reaction better than other NTPs. The turnover number for the single-stranded DNA-stimulated dATPase activity was 1380 min(-1), approximately 1.5-fold higher than that observed for the ATPase activity (900 min(-1)). The purified protein catalyzed unwinding of partial duplex substrates up to at least 93 bp, however, unwinding of an 89 bp blunt duplex substrate was not detected.

Biochemical characterization of the equine arteritis virus helicase suggests a close functional relationship between arterivirus and coronavirus helicases.

The arterivirus equine arteritis virus nonstructural protein 10 (nsp10) has previously been predicted to contain a Zn finger structure linked to a superfamily 1 (SF1) helicase domain. A recombinant form of nsp10, MBP-nsp10, was produced in Escherichia coli as a fusion protein with the maltose-binding protein. The protein was partially purified by affinity chromatography and shown to have ATPase activity that was strongly stimulated by poly(dT), poly(U), and poly(dA) but not by poly(G). The protein also had both RNA and DNA duplex-unwinding activities that required the presence of 5' single-stranded regions on the partial-duplex substrates, indicating a 5'-to-3' polarity in the unwinding reaction. Results of this study suggest a close functional relationship between the arterivirus nsp10 and the coronavirus helicase, for which NTPase and duplex-unwinding activities were recently demonstrated. In a number of biochemical properties, both arterivirus and coronavirus SF1 helicases differ significantly from the previously characterized RNA virus SF1 and SF2 enzymes. Thus, the combined data strongly support the idea that nidovirus helicases may represent a separate group of RNA virus-encoded helicases with distinct properties.

The human coronavirus 229E superfamily 1 helicase has RNA and DNA duplex-unwinding activities with 5'-to-3' polarity.

The human coronavirus 229E replicase gene encodes a protein, p66HEL, that contains a putative zinc finger structure linked to a putative superfamily (SF) 1 helicase. A histidine-tagged form of this protein, HEL, was expressed using baculovirus vectors in insect cells. The purified recombinant protein had in vitro ATPase activity that was strongly stimulated by poly(U), poly(dT), poly(C), and poly(dA), but not by poly(G). The recombinant protein also had both RNA and DNA duplex-unwinding activities with 5'-to-3' polarity. The DNA helicase activity of the enzyme preferentially unwound 5'-oligopyrimidine-tailed, partial-duplex substrates and required a tail length of at least 10 nucleotides for effective unwinding. The combined data suggest that the coronaviral SF1 helicase functionally differs from the previously characterized RNA virus SF2 helicases.

Initiation of DNA replication by simian virus 40 T antigen is inhibited by the p107 protein.

The p107 protein is related to the Rb protein by a 60-kDa region of homology called the pocket domain that binds cellular proteins such as E2F and cyclins A and E as well as the viral oncoproteins E1A, E7, and SV40 T antigen. The p107 and Rb proteins have both been implicated as negative regulators of cell growth. We have examined the effect of the unphosphorylated pocket domain of p107 on specific stages of the T antigen-mediated replication of SV40 DNA in vitro. The pocket domain inhibited replication by preventing the assembly of T antigen at the SV40 core origin DNA containing binding site II. However, both proteins formed a ternary complex with DNA containing T antigen-binding site I. The pocket domain of p107 did not inhibit the oligomerization of T antigen in the absence of DNA, and the p107 derivative is bound to the intermediates of this reaction. In the unwinding assay, once T antigen was preassembled as hexamers at the core origin, the pocket domain bound to and stabilized the complex, resulting in an increase in the yield of unwound product. Preformed T antigen hexamers complexed with the pocket domain bind to a synthetic replication fork, and this complex supported unwinding. Consistent with this, the p107 pocket domain had no effect on the helicase activity of T antigen in an assay using a partial duplex substrate. However, a complex containing p107 and T antigen assembled at the core origin did not support SV40 DNA replication in HeLa cell crude extracts or in the monopolymerase reaction. This inhibition is due to the inability of the complex to bind to DNA polymerase alpha, which is required for the initiation of DNA synthesis. The data suggest pathways by which the pocket domain of p107 can negatively regulate T antigen-mediated replication in vitro. The binding of T antigen to p107 is discussed with respect to its role in mitigating negative cell growth control, resulting in viral mediated transformation.

Purification and characterization of a DNA helicase from Saccharomyces cerevisiae.

A novel DNA helicase, scHelI, has been purified from whole cell extracts of Saccharomyces cerevisiae using biochemical assays to monitor the fractionation. The enzyme unwinds partial duplex DNA substrates, as long as 343 base pairs in length, in a reaction that is dependent on either ATP or dATP hydrolysis. scHelI also catalyzes a single-stranded DNA-dependent ATP hydrolysis reaction; the apparent Km for ATP is 325 microM. The unwinding reaction on circular partial duplex substrates is biphasic, with a fast component occurring within 5 min of the initiation of the reaction and a slow component continuing to 60 min. This is in contrast to the ATP hydrolysis reaction, which exhibits linear kinetics for 60 min. The direction of the unwinding reaction is 5' to 3' with respect to the strand of DNA on which the enzyme is bound. The unwinding reaction is strongly stimulated by the addition of Escherichia coli single-stranded DNA-binding protein when long partial duplex substrates are used. The enzymatic activity of scHelI copurifies with a polypeptide of 135 kDa as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The polypeptide sediments as a monomer in a glycerol gradient in the presence of 0.2 M NaCl.