Nucleic Acid-dependent ATPase Research Articles

Monitoring functional RNA binding of RNA-dependent ATPase enzymes such as SF2 helicases using RNA dependent ATPase assays: A RIG-I case study.

The highly conserved Superfamily 1 (SF1) and Superfamily 2 (SF2) nucleic acid-dependent ATPases, are ubiquitous motor proteins with central roles in DNA and RNA metabolism (Jankowsky & Fairman, 2007). These enzymes require RNA or DNA binding to stimulate ATPase activity, and the conformational changes that result from this coupled behavior are linked to a multitude of processes that range from nucleic acid unwinding to the flipping of macromolecular switches (Pyle, 2008, 2011). Knowledge about the relative affinity of nucleic acid ligands is crucial for deducing mechanism and understanding biological function of these enzymes. Because enzymatic ATPase activity is directly coupled to RNA binding in these proteins, one can utilize their ATPase activity as a simple reporter system for monitoring functional binding of RNA or DNA to an SF1 or SF2 enzyme. In this way, one can rapidly assess the relative impact of mutations in the protein or the nucleic acid and obtain parameters that are useful for setting up more quantitative direct binding assays. Here, we describe a routine method for employing NADH-coupled enzymatic ATPase activity to obtain kinetic parameters reflecting apparent ATP and RNA binding to an SF2 helicase. First, we provide a protocol for calibrating an NADH-couple ATPase assay using the well-characterized ATPase enzyme hexokinase, which a simple ATPase enzyme that is not coupled with nucleic acid binding. We then provide a protocol for obtaining kinetic parameters (KmATP, Vmax and KmRNA) for an RNA-coupled ATPase enzyme, using the double-stranded RNA binding protein RIG-I as a case-study. These approaches are designed to provide investigators with a simple, rapid method for monitoring apparent RNA association with SF2 or SF1 helicases.

Single-molecule studies of helicases and translocases in prokaryotic genome-maintenance pathways

Helicases involved in genomic maintenance are a class of nucleic-acid dependent ATPases that convert the energy of ATP hydrolysis into physical work to execute irreversible steps in DNA replication, repair, and recombination. Prokaryotic helicases provide simple models to understand broadly conserved molecular mechanisms involved in manipulating nucleic acids during genome maintenance. Our understanding of the catalytic properties, mechanisms of regulation, and roles of prokaryotic helicases in DNA metabolism has been assembled through a combination of genetic, biochemical, and structural methods, further refined by single-molecule approaches. Together, these investigations have constructed a framework for understanding the mechanisms that maintain genomic integrity in cells. This review discusses recent single-molecule insights into molecular mechanisms of prokaryotic helicases and translocases.

Structural insights into the inhibition of bacterial RecA by naphthalene polysulfonated compounds.

SummaryAs a promising target for alternative antimicrobials, bacterial recombinase A (RecA) protein has attracted much attention for its roles in antibiotic-driven SOS response and mutagenesis. Naphthalene polysulfonated compounds (NPS) such as suramin have previously been explored as antibiotic adjuvants targeting RecA, although the underlying structural bases for RecA-ligand interactions remain obscure. Based on our in silico predictions and documented activity of NPS in vitro, we conclude that the analyzed NPS likely interact with Tyr103 (Y103) and other key residues in the ATPase activity center (pocket A). For validation, we generated recombinant RecA proteins (wild-type versus Y103 mutant) to determine the binding affinities for RecA protein interactions with suramin and underexamined NPS in isothermal titration calorimetry. The corresponding dissociation constants (Kd) ranged from 11.5 to 18.8 μM, and Y103 was experimentally shown to be critical to RecA-NPS interactions.

Purification and Characterization of Double-Stranded Nucleic Acid-Dependent ATPase Activities of Tagged Dicer-Related Helicase 1 and its Short Isoform in Caenorhabditis elegans.

The Dicer-related helicases (DRHs) are members of a helicase subfamily, and mammalian DRHs such as retinoic acid-inducible gene-I (RIG-I), are involved in antiviral immunity. Caenorhabditis elegans DRH-1 and DRH-3 play crucial roles in antiviral function and chromosome segregation, respectively. Although intrinsic double-stranded RNA-dependent ATP-hydrolyzing activity has been observed in the recombinant DRH-3 protein prepared from Escherichia coli, there are no reports of biochemical studies of the nematode RIG-I homolog DRH-1. In this study, the secondary structure prediction by JPred4 revealed that DRH-1 and DRH-3 had distinct N-terminal regions and that a 200-amino acid N-terminal region of DRH-1 could form a structure very rich in α-helices. We investigated expressions and purifications of a codon-optimized DRH-1 with four different N-terminal tags, identifying poly-histidine (His)-small ubiquitin-like modifier (SUMO) as a suitable tag for DRH-1 preparation. Full-length (isoform a) and a N-terminal truncated (isoform b) of DRH-1 were purified as the His-SUMO-tagged fusion proteins. Finally, the nucleic acid-dependent ATPase activities were investigated for the two His-SUMO-tagged DRH-1 isoforms and His-tagged DRH-3. The tagged DRH-3 exhibited dsRNA-dependent ATPase activity. However, detectable dsRNA dependency of ATPase activities was not found in either isoform of tagged DRH-1 and a tag-free DRH-1 (isoform a) treated with SUMO protease. These observations suggest that DRH-1 and its short isoform have no or poor nucleic acid-dependent ATPase activity, unlike DRH-3 and mammalian DRHs.

High-Throughput Screening to Identify Inhibitors of DEAD Box Helicase DDX41

The human DEAD (Asp–Glu–Ala–Asp) box protein DDX41, a member of the DEXDc helicase family, has nucleic acid–dependent ATPase and RNA and DNA translocase and unwinding activities. DDX41 is affected by somatic mutations in sporadic cases of myeloid neoplasms as well as in a biallelic fashion in 50% of patients with germline DDX41 mutations. The R525H mutation in DDX41 is thought to play important roles in the development of hereditary myelodysplastic syndrome and acute myelocytic leukemia. In this study, human DDX41 and its R525H mutant (R525H) were expressed in Escherichia coli and purified. The ATPase activities of the recombinant DDX41 and R525H proteins were dependent on both ATP and double-stranded DNA (dsDNA), such as poly(dG–dC) and poly(dA–dT). High-throughput screening was performed with a dsDNA-dependent ATPase assay using the human R525H proteins. After hit confirmation and counterscreening, several small-molecule inhibitors were successfully identified. These compounds show DDX41-selective inhibitory activities.

Structure of the DEAH/RHA ATPase Prp43p bound to RNA implicates a pair of hairpins and motif Va in translocation along RNA.

Three families of nucleic acid-dependent ATPases (DEAH/RHA, Ski2-like, and NS3/NPH-II), termed the DExH ATPases, are thought to execute myriad functions by processive, ATP-dependent, 3′ to 5′ translocation along single-stranded nucleic acid. While the mechanism of translocation of the viral NS3/NPH-II family has been studied extensively, it has not been clear if or how the principles that have emerged for this family extend to the other two families. Here we report the crystal structure of the yeast DEAH/RHA family ATPase Prp43p, which functions in splicing and ribosome biogenesis, in complex with poly-uracil and a nonhydrolyzable ATP analog. The structure reveals a conserved DEAH/RHA-specific variation of motif Ib within the RecA1 domain of the catalytic core, in which the motif elongates as a β-hairpin that bookends the 3′ end of a central RNA stack, a function that in the viral and Ski-2 families is performed by an auxiliary domain. Supporting a fundamental role in translocation, mutations in this hairpin abolished helicase activity without affecting RNA binding or ATPase activity. While the structure reveals differences with viral ATPases in the RecA1 domain, our structure demonstrates striking similarities with viral ATPases in the RecA2 domain of the catalytic core, including both a prominent β-hairpin that bookends the 5′ end of the RNA stack and a dynamic motif Va that is implicated in mediating translocation. Our crystal structure, genetic, and biochemical experiments, as well as comparisons with other DExH ATPases, support a generalized mechanism for the DExH class of helicases involving a pair of bookends that inchworm along RNA.

Probing Single Helicase Dynamics on Long Nucleic Acids Through Fluorescence-Force Measurement.

Helicases are nucleic acid-dependent ATPases which can bind and remodel nucleic acids, protein-nucleic acid complexes, or both. They are involved in almost every step in cells related to nucleic acid metabolisms, including DNA replication and repair, transcription, RNA maturation and splicing, and nuclear export processes. Using single-molecule fluorescence-force spectroscopy, we have previously directly observed helicase translocation on long single-stranded DNA and revealed that two monomers of UvrD helicase are required for the initiation of unwinding function. Here, we present the details of fluorescence-force spectroscopy instrumentation, calibration, and activity assays in detail for observing the biochemical activities of helicases in real time and revealing how mechanical forces are involved in protein-nucleic acid interaction. These single-molecule approaches are generally applicable to many other protein-nucleic acid systems.

Double-stranded DNA-dependent ATPase Irc3p is directly involved in mitochondrial genome maintenance.

Nucleic acid-dependent ATPases are involved in nearly all aspects of DNA and RNA metabolism. Previous studies have described a number of mitochondrial helicases. However, double-stranded DNA-dependent ATPases, including translocases or enzymes remodeling DNA-protein complexes, have not been identified in mitochondria of the yeast Saccharomyces cerevisae. Here, we demonstrate that Irc3p is a mitochondrial double-stranded DNA-dependent ATPase of the Superfamily II. In contrast to the other mitochondrial Superfamily II enzymes Mss116p, Suv3p and Mrh4p, which are RNA helicases, Irc3p has a direct role in mitochondrial DNA (mtDNA) maintenance. Specific Irc3p-dependent mtDNA metabolic intermediates can be detected, including high levels of double-stranded DNA breaks that accumulate in irc3Δ mutants. irc3Δ-related topology changes in rho- mtDNA can be reversed by the deletion of mitochondrial RNA polymerase RPO41, suggesting that Irc3p counterbalances adverse effects of transcription on mitochondrial genome stability.

Mycobacterium smegmatis Lhr Is a DNA-dependent ATPase and a 3′-to-5′ DNA Translocase and Helicase That Prefers to Unwind 3′-Tailed RNA:DNA Hybrids

We are interested in the distinctive roster of helicases of Mycobacterium, a genus of the phylum Actinobacteria that includes the human pathogen Mycobacterium tuberculosis and its avirulent relative Mycobacterium smegmatis. Here, we identify and characterize M. smegmatis Lhr as the exemplar of a novel clade of superfamily II helicases, by virtue of its biochemical specificities and signature domain organization. Lhr is a 1507-amino acid monomeric nucleic acid-dependent ATPase that uses the energy of ATP hydrolysis to drive unidirectional 3'-to-5' translocation along single strand DNA and to unwind duplexes en route. The ATPase is more active in the presence of calcium than magnesium. ATP hydrolysis is triggered by either single strand DNA or single strand RNA, yet the apparent affinity for a DNA activator is 11-fold higher than for an RNA strand of identical size and nucleobase sequence. Lhr is 8-fold better at unwinding an RNA:DNA hybrid than it is at displacing a DNA:DNA duplex of identical nucleobase sequence. The truncated derivative Lhr-(1-856) is an autonomous ATPase, 3'-to-5' translocase, and RNA:DNA helicase. Lhr-(1-856) is 100-fold better RNA:DNA helicase than DNA:DNA helicase. Lhr homologs are found in bacteria representing eight different phyla, being especially prevalent in Actinobacteria (including M. tuberculosis) and Proteobacteria (including Escherichia coli).

Plasmodium falciparum DOZI, an RNA helicase interacts with eIF4E

DEAD box RNA helicases play crucial roles in RNA metabolism such as splicing, ribosome biogenesis, RNA transport, degradation and translation. DDX6/DOZI (development of zygote inhibited) is one of the well characterized member of the DEAD box family and is highly conserved from humans to malaria parasite. DDX6 is involved in a variety of biological processes, which include the sexual development of the protozoan parasite. In the present manuscript we report that P. falciparum DOZI (DDX6 homologue); PfDZ50 contains the characteristic DNA and RNA binding, nucleic acid-dependent ATPase and RNA unwinding activities. Enzymatic characterization of truncated derivatives of PfDZ50 such as PfDZ50T1 (domain 1) and PfDZ50T2 (domain 2) shows that none of them contains ATPase activity. Furthermore, we confirmed that PfDZ50 interacts with PfeIF4E mainly through domain 1. Using in vitro translation assays we show that PfDZ50 inhibits translation. With the same assays we further report that externally added PfeIF4E restores ~70% of translation. Using immunofluorescence assays we demonstrate that PfDZ50 is localized mainly in the cytoplasm in the asexual intraerythrocytic developmental stages of P. falciparum. The localization pattern further suggests that PfDZ50 appears typically in granular bodies throughout the cytoplasm. Thus these studies will advance our knowledge regarding the function of PfDZ50/DDX6 in general.

Human UPF1 interacts with TPP1 and telomerase and sustains telomere leading-strand replication

Eukaryotic up-frameshift 1 (UPF1) is a nucleic acid-dependent ATPase and 5'-to-3' helicase, best characterized for its roles in cytoplasmic RNA quality control. We previously demonstrated that human UPF1 binds to telomeres in vivo and its depletion leads to telomere instability. Here, we show that UPF1 is present at telomeres at least during S and G2/M phases and that UPF1 association with telomeres is stimulated by the phosphoinositide 3-kinase (PI3K)-related protein kinase ataxia telangiectasia mutated and Rad3-related (ATR) and by telomere elongation. UPF1 physically interacts with the telomeric factor TPP1 and with telomerase. Akin to UPF1 binding to telomeres, this latter interaction is mediated by ATR. Moreover, the ATPase activity of UPF1 is required to prevent the telomeric defects observed upon UPF1 depletion, and these defects stem predominantly from inefficient telomere leading-strand replication. Our results portray a scenario where UPF1 orchestrates crucial aspects of telomere biology, including telomere replication and telomere length homeostasis.

A Motif Unique to the Human Dead-Box Protein DDX3 Is Important for Nucleic Acid Binding, ATP Hydrolysis, RNA/DNA Unwinding and HIV-1 Replication

DEAD-box proteins are enzymes endowed with nucleic acid-dependent ATPase, RNA translocase and unwinding activities. The human DEAD-box protein DDX3 has been shown to play important roles in tumor proliferation and viral infections. In particular, DDX3 has been identified as an essential cofactor for HIV-1 replication. Here we characterized a set of DDX3 mutants biochemically with respect to nucleic acid binding, ATPase and helicase activity. In particular, we addressed the functional role of a unique insertion between motifs I and Ia of DDX3 and provide evidence for its implication in nucleic acid binding and HIV-1 replication. We show that human DDX3 lacking this domain binds HIV-1 RNA with lower affinity. Furthermore, a specific peptide ligand for this insertion selected by phage display interferes with HIV-1 replication after transduction into HelaP4 cells. Besides broadening our understanding of the structure-function relationships of this important protein, our results identify a specific domain of DDX3 which may be suited as target for antiviral drugs designed to inhibit cellular cofactors for HIV-1 replication.

Inhibition of unwinding and ATPase activities of Plasmodium falciparum Dbp5/DDX19 homologue

We have recently reported the isolation and characterization of Plasmodium falciparum Dbp5/DDX19 homologue PfD66 and the results indicate that it contains ATP-dependent bipolar DNA and RNA unwinding activity, intrinsic nucleic acid-dependent ATPase and RNA-binding activities (Molecular and Biochemical Parasitology, (In press) 10.1016/j.molbiopara.2010.12.003). In the present study we report the effect of a number of compounds such as actinomycin D, aphidicolin, camptothecin, cyclophosphamide, 4’,6’-di-amidino-2-phenylindole (DAPI), daunorubicin, distamycin, ethidium bromide, ellipticine, genistein, mitoxantrone, nalidixic acid, netropsin, nogalamycin, novobiocin and VP-16 on the DNA unwinding and ATPase activities of PfD66. The results indicate that DAPI, ethidium bromide, netropsin and nogalamycin efficiently inhibited the helicase and ATPase activities of PfD66. These studies will make an important contribution in understanding the mechanism of DNA unwinding by Plasmodium falciparum helicase PfD66.

A novel dual Dbp5/DDX19 homologue from Plasmodium falciparum requires Q motif for activity

Helicases are ubiquitous essential enzymes which have significant role in the nucleic acid metabolism. Using in silico approaches in the recent past we have identified a number of helicases in the Plasmodium falciparum genome. In the present study we report purification and detailed characterization of a novel helicase from P. falciparum. Our results indicate that this helicase is a homologue of Dbp5 and DDX19 from yeast and human, respectively. The biochemical characterization shows that it contains DNA and RNA unwinding, nucleic acid dependent ATPase and RNA binding activities. It is interesting to note that this enzyme can unwind DNA duplexes in both 5′ to 3′ and 3′ to 5′ directions. Using truncated derivatives we further show that Q motif is essentially required for all of its activities. These studies should make an important contribution in understanding the enzymes involved in nucleic acid metabolism in the parasite.

A novel DEAD box helicase Has1p from Plasmodium falciparum: N-terminal is essential for activity

Helicases catalyze the opening of nucleic acid duplexes and are implicated in many nucleic acid metabolic cellular processes that require single stranded DNA or reorganization of RNA structure. Previously we have reported that Plasmodium falciparum genome contains a number of DEAD box helicases. In the present study we report the cloning, expression and characterization of one of the novel members of DEAD box family from P. falciparum. Our results indicate that it is a homologue of Has1p from yeast and it contains DNA and RNA unwinding, nucleic acid-dependent ATPase and RNA binding activities. This enzyme can utilize all the nucleosidetriphosphates (NTPs) and deoxy nucleosidetriphosphates (dNTPs) for its unwinding activity. Using a truncated derivative of this protein we further report that the N-terminal region of the protein is essentially required for its activity. These studies suggest that besides the conserved helicase domain the highly variable N-terminal region also contributes in the activity of the protein.

Characterization of replication fork and phosphorylation stimulated Plasmodium falciparum helicase 45

Helicases are essential enzymes, which play important role in the metabolism of nucleic acids. In the present study we report further characterization of PfH45 ( Plasmodium falciparum helicase 45), which is an essential enzyme for parasite survival. The results show that the helicase activity of PfH45 is significantly stimulated by replication fork like structure. The studies using truncated derivatives of PfH45 show that its nucleic acid dependent ATPase activity resides in the N-terminal one third of the protein and its RNA and DNA-binding activity predominantly resides in the C-terminal two third of the protein. The phosphorylation of PfH45 by protein kinase C at Ser and Thr residues stimulated its DNA and RNA helicase and ssDNA and RNA-dependent ATPase activities. DNA-interacting compounds actinomycin, DAPI, daunorubicin, ethidium bromide, netropsin and nogalamycin were able to inhibit the helicase and ssDNA-dependent ATPase activity with apparent IC 50 values ranging from 0.5 to 5.0 μM respectively. These compounds distinctively inhibit the helicase activity probably by forming complex with DNA and obstructing enzyme movement.

The nucleoside triphosphatase and helicase activities of vaccinia virus NPH-II are essential for virus replication.

Vaccinia virus NPH-II is the prototypal RNA helicase of the DExH box protein family, which is defined by six shared sequence motifs. The contributions of conserved amino acids in motifs I (TGVGKTSQ), Ia (PRI), II (DExHE), and III (TAT) to enzyme activity were assessed by alanine scanning. NPH-II-Ala proteins were expressed in baculovirus-infected Sf9 cells, purified, and characterized with respect to their RNA helicase, nucleic acid-dependent ATPase, and RNA binding functions. Alanine substitutions at Lys-191 and Thr-192 (motif I), Arg-229 (motif Ia), and Glu-300 (motif II) caused severe defects in RNA unwinding that correlated with reduced rates of ATP hydrolysis. In contrast, alanine mutations at His-299 (motif II) and at Thr-326 and Thr-328 (motif III) elicited defects in RNA unwinding but spared the ATPase. None of the mutations analyzed affected the binding of NPH-II to RNA. These findings, together with previous mutational studies, indicate that NPH-II motifs I, Ia, II, and VI (QRxGRxGRxxxG) are essential for nucleoside triphosphate (NTP) hydrolysis, whereas motif III and the His moiety of the DExH-box serve to couple the NTPase and helicase activities. Wild-type and mutant NPH-II-Ala genes were tested for the ability to rescue temperature-sensitive nph2-ts viruses. NPH-II mutations that inactivated the phosphohydrolase in vitro were lethal in vivo, as judged by the failure to recover rescued viruses containing the Ala substitution. The NTPase activity was necessary, but not sufficient, to sustain virus replication, insofar as mutants for which NTPase was uncoupled from unwinding (H299A, T326A, and T328A) were also lethal. We conclude that the phosphohydrolase and helicase activities of NPH-II are essential for virus replication.

The QRxGRxGRxxxG motif of the vaccinia virus DExH box RNA helicase NPH-II is required for ATP hydrolysis and RNA unwinding but not for RNA binding

Vaccinia virus NPH-II is an essential nucleic acid-dependent nucleoside triphosphate that catalyzes unidirectional unwinding of duplex RNA containing a 3' tail. NPH-II is the prototypal RNA helicase of the DExH box protein family, which is defined by several shared sequence motifs. The contribution of the conserved QRKGRVGRVNPG region to enzyme activity was assessed by alanine-scanning mutagenesis. Ten mutated versions of NPH-II were expressed in vaccinia virus-infected BSC-40 cells and purified by nickel affinity chromatography and glycerol gradient sedimentation. The mutated proteins were characterized with respect to RNA helicase, nucleic acid-dependent ATPase, and RNA binding functions. Individual alanine substitutions at invariant residues Q-491, G-494, R-495, G-497, R-498, and G-502 caused severe defects in RNA unwinding that correlated with reduced rates of ATP hydrolysis. None of these mutations affected the binding of NPH-II to single-strand RNA or to the tailed duplex RNA used as a helicase substrate. Mutation of the strictly conserved position R-492 inhibited ATPase and helicase activities and also caused a modest decrement in RNA binding. Alanine mutations at the nonconserved position N-500 and the weakly conserved residue P-501 had no apparent effect on any activity associated with NPH-II, whereas a mutation at the weakly conserved position K-493 reduced helicase to one-third and ATPase to two-thirds of the activity of wild-type required for ATP hydrolysis and RNA unwinding but not for RNA binding. Because mutations in the HRxGRxxR motif of the prototypal DEAD box RNA helicase eIF-4A abolish or severely inhibit RNA binding, we surmise that the contribution of conserved helicase motifs to overall protein function is context dependent.

Vaccinia virion protein I8R has both DNA and RNA helicase activities: implications for vaccinia virus transcription

A nucleic acid-dependent ATPase was purified from vaccinia virions and shown to have both DNA:DNA and RNA:RNA helicase activities. This is only the third helicase to be identified that can unwind both DNA and RNA duplexes. The DNA helicase activity copurified with nucleoside triphosphate phosphohydrolase II (NPHII), an RNA helicase encoded by gene I8R (S. Shuman, Proc. Natl. Acad. Sci. USA 89:10935-10939, 1992). Immunodepletion with two antisera to NPHII and analysis of recombinant NPHII protein (C. H. Gross and S. Shuman, J. Virol. 69:4727-4736, 1995) confirmed that the DNA helicase activity was encoded by the I8R gene. The I8R DNA helicase unwound DNA in a 3'-to-5' direction only, unwound duplexes of 35 bp but not 45 bp, and could be stimulated to unwind longer duplexes by the Escherichia coli single-stranded DNA-binding protein. DNA helicase activity was not stimulated by salt and was sensitive to 100 mM NaCl or KCl. The I8R protein has amino acid similarity to human RNA helicase A and to nuclear DNA helicase II, a bovine DNA and RNA helicase. On the basis of the phenotype of I8R temperature-sensitive mutants, it was suggested that the I8R protein is not required for DNA replication but might aid in the extrusion of early mRNA from the virus core. The DNA helicase activity of the I8R protein allows another interpretation of the mutant phenotype, namely, that the I8R DNA helicase activity is required for initiation of early transcription from within vaccinia virions.

Suppression of Mutations in Two Saccharomyces cerevisiae Genes by the Adenovirus E1A Protein

The protein products of the adenoviral E1A gene are implicated in a variety of transcriptional and cell cycle events, involving interactions with several proteins present in human cells, including parts of the transcriptional machinery and negative regulators of cell division such as the Rb gene product and p107. To determine if there are functional homologs of E1A in Saccharomyces cerevisiae, we have developed a genetic screen for mutants that depend on E1A for growth. The screen is based on a colony color sectoring assay which allows the identification of mutants dependent on the maintenance and expression of an E1A-containing plasmid. Using this screen, we have isolated five mutants that depend on expression of the 12S or 13S cDNA of E1A for growth. A plasmid shuffle assay confirms that the plasmid-dependent phenotype is due to the presence of either the 12S or the 13S E1A cDNA and that both forms of E1A rescue growth of all mutants equally well. The five mutants fall into two classes that were named web1 and web2 (for "wants E1A badly"). Plasmid shuffle assays with mutant forms of E1A show that conserved region 1 (CR1) is required for rescue of the growth of the web1 and web2 E1A-dependent yeast mutants, while the N-terminal 22 amino acids are only partially required; conserved region 2 (CR2) and the C terminus are dispensable. The phenotypes of mutants in both the web1 and the web2 groups are due to a single gene defect, and the yeast genes that fully complement the mutant phenotypes of both groups were cloned. The WEB1 gene sequence encodes a 1,273-amino-acid protein that is identical to SEC31, a protein involved in the budding of transport vesicles from the endoplasmic reticulum. The WEB2 gene encodes a 1,522-amino-acid protein with homology to nucleic acid-dependent ATPases. Deletion of either WEB1 or WEB2 is lethal. Expression of E1A is not able to rescue the lethality of either the web1 or the web2 null allele, implying allele-specific mutations that lead to E1A dependence.