Bipartite Binding Research Articles

Uncharged tRNA Activates GCN2 by Displacing the Protein Kinase Moiety from a Bipartite tRNA-Binding Domain

Protein kinase GCN2 regulates translation in amino acid–starved cells by phosphorylating eIF2. GCN2 contains a regulatory domain related to histidyl-tRNA synthetase (HisRS) postulated to bind multiple deacylated tRNAs as a general sensor of starvation. In accordance with this model, GCN2 bound several deacylated tRNAs with similar affinities, and aminoacylation of tRNA Phe weakened its interaction with GCN2. Unexpectedly, the C-terminal ribosome binding segment of GCN2 (C-term) was required in addition to the HisRS domain for strong tRNA binding. A combined HisRS+C-term segment bound to the isolated protein kinase (PK) domain in vitro, and tRNA impeded this interaction. An activating mutation ( GCN2 c-E803V ) that weakens PK–C-term association greatly enhanced tRNA binding by GCN2. These results provide strong evidence that tRNA stimulates the GCN2 kinase moiety by preventing an inhibitory interaction with the bipartite tRNA binding domain.

Region-specific activation of the Xenopus brachyury promoter involves active repression in ectoderm and endoderm: a study using transgenic frog embryos.

Tissue specification in the early embryo requires the integration of spatial information at the promoters of developmentally important genes. Although several response elements for signalling pathways have been identified in Xenopus promoters, it is not yet understood what defines the sharp borders that restrict expression to a specific tissue. Here we use transgenic frog embryos to study the spatial and temporal regulation of the Xbra promoter. Deletion analysis and point mutations in putative transcription factor-binding sites identified two repressor modules, which exert their main effects at different stages during gastrulation. One module is defined by a bipartite binding site for a Smad-interacting protein (SIP1) of the deltaEF1 repressor family and acts to confine expression to the marginal zone early in gastrulation. The other module is defined by two homeodomain-binding sites and is responsible for repression in dorsal mesoderm and ectoderm at mid-gastrula stages. In addition, an upstream region of the promoter is necessary to repress expression in neural tissues later in development. Together, our results show that repression plays an important role in the restriction of Xbra expression to the mesoderm, and we suggest that similar mechanisms may be involved in the spatial regulation of other genes in early embryonic development.

Paired-homeodomain transcription factor PAX4 acts as a transcriptional repressor in early pancreatic development.

The paired-homeodomain transcription factor PAX4 is expressed in the developing pancreas and along with PAX6 is required for normal development of the endocrine cells. In the absence of PAX4, the numbers of insulin-producing beta cells and somatostatin-producing delta cells are drastically reduced, while the numbers of glucagon-producing alpha cells are increased. To gain insight into PAX4 function, we cloned a full-length Pax4 cDNA from a beta-cell cDNA library and identified a bipartite consensus DNA binding sequence consisting of a homeodomain binding site separated from a paired domain binding site by 15 nucleotides. The paired half of this consensus sequence has similarities to the PAX6 paired domain consensus binding site, and the two proteins bind to common sequences in several islet genes, although with different relative affinities. When expressed in an alpha-cell line, PAX4 represses transcription through the glucagon or insulin promoters or through an isolated PAX4 binding site. This repression is not simply due to competition with the PAX6 transcriptional activator for the same binding site, since PAX4 fused to the unrelated yeast GAL4 DNA binding domain also represses transcription through the GAL4 binding site in the alpha-cell line and to a lesser degree in beta-cell lines and NIH 3T3 cells. Repressor activity maps to more than one domain within the molecule, although the homeodomain and carboxyl terminus give the strongest repression. PAX4 transcriptional regulation apparently plays a role only early in islet development, since Pax4 mRNA as determined by reverse transcriptase PCR peaks at embryonic day 13.5 in the fetal mouse pancreas and is undetectable in adult islets. In summary, PAX4 can function as a transcriptional repressor and is expressed early in pancreatic development, which may allow it to suppress alpha-cell differentiation and permit beta-cell differentiation.

Ligand-induced Structural Alterations in Human Iron Regulatory Protein-1 Revealed by Protein Footprinting

Human iron regulatory protein-1 (IRP-1) is a bifunctional protein that regulates iron metabolism by binding to mRNAs encoding proteins involved in iron uptake, storage, and utilization. Intracellular iron accumulation regulates IRP-1 function by promoting the assembly of an iron-sulfur cluster, conferring aconitase activity to IRP-1, and hindering RNA binding. Using protein footprinting, we have studied the structure of the two functional forms of IRP-1 and have mapped the surface of the iron-responsive element (IRE) binding site. Binding of the ferritin IRE or of the minimal regulatory region of transferrin receptor mRNA induced strong protections against proteolysis in the region spanning amino acids 80 to 187, which are located in the putative cleft thought to be involved in RNA binding. In addition, IRE-induced protections were also found in the C-terminal domain at Arg-721 and Arg-728. These data implicate a bipartite IRE binding site located in the putative cleft of IRP-1. The aconitase form of IRP-1 adopts a more compact structure because strong reductions of cleavage were detected in two defined areas encompassing residues 149 to 187 and 721 to 735. Thus both ligands of apo-IRP-1, the IRE and the 4Fe-4S cluster, induce distinct but overlapping alterations in protease accessibility. These data provide evidences for structural changes in IRP-1 upon cluster formation that affect the accessibility of residues constituting the RNA binding site.

A new model of cell cycle-regulated transcription: repression of the cyclin A promoter by CDF-1 and anti-repression by E2F.

Cell cycle regulation of the cyclin A gene is determined by a bipartite repressor binding site in the region of the basal promoter, termed CDE-CHR, which also controls the expression of cell cycle genes upregulated in S or G2 (such as cdc25C). The CDE-CHR in the cyclin A promoter is recognized by both E2F complexes and CDF-1, but the contribution of each of these factors in cell cycle regulation is unknown. In the present study, we have introduced mutations into the cyclin A promoter which lead to either a loss or enhancement of E2F binding, while having only marginal effects on the interaction with CDF-1. Unlike mutants deficient for CDF-1 binding, promoter variants lacking E2F binding showed an unchanged repression in G0, thus identifying CDF-1 as the principal repressor of the cyclin A gene. The same mutants did show, however, a delayed derepression while a mutation leading to increased E2F binding resulted in premature up-regulation. These findings clearly suggest that E2F contributes to the correct timing of cyclin A transcription, presumably by acting as an anti-repressor. In agreement with this conclusion, we find that the cyclin A promoter only poorly interacts with E2F-4, which is the major E2F family member in G0 cells, while a clear binding is seen with E2F-1 and -3, which are up-regulated in late G1.

POU domain factors in neural development.

Transcription factors serve critical roles in the progressive development of general body plan, organ commitment, and finally, specific cell types. Comparison of the biological roles of a series of individual members within a family permits some generalizations to be made regarding the developmental events that are likely to be regulated by a particular class of transcription factors. Here, we evidence that the developmental functions of the family of transcription factors characterized by the POU DNA binding motif exerts roles in mammalian development. The POU domain family of transcription factors was defined following the observation that the products of three mammalian genes, Pit-1, Oct-1, and Oct-2, and the protein encoded by the C. elegans gene unc-86, shared a region of homology, known as the POU domain. The POU domain is a bipartite DNA binding domain, consisting of two highly conserved regions, tethered by a variable linker. The approximately 75 amino acid N-terminal region was called the POU-specific domain and the C-terminal 60 amino acid region, the POU-homeodomain. High-affinity site-specific DNA binding by POU domain transcription factors requires both the POU-specific and the POU-homeodomain. Resolution of the crystal structures of Oct-1 and Pit-1 POU domains bound to DNA as a monomer and homodimer, respectively, confirmed several of the in vitro findings regarding interactions of this bipartite DNA binding domain with DNA and has provided important information regarding the flexibility and versatility of POU domain proteins. Overall the crystal structure of a monomer of the Oct-1 POU domain bound to the octamer element was similar to that predicted by the NMR solution structures of the POU-specific domain and the POU-homeodomain in isolation, with the POU-specific domain consists of four alpha helices, with the second and third helices forming a structure similar to the helix-turn-helix motif of the lambda and 434 repressors; several of the DNA base contacts are also conserved. A homodimer of the Pit-1 POU domain was crystallized bound to a Pit-1 dimer DNA element that is closely related to a site in the proximal promoter of the prolactin gene. The structure of the Pit-1 POU domain on DNA is very similar to that of Oct-1, and the Pit-1 POU-homeodomain/DNA structure is strikingly similar to that of other homeodomains, including the Oct-1 POU-homeodomain. The DNA contacts made by the Pit-1 POU-specific domain are also similar to those of Oct-1 and conserved with many made by the prokaryotic repressors. In the Oct-1 crystal, the POU-specific domain recognizes a GCAT half-site, while the corresponding sequence recognized by the Pit-1 POU-specific domain, GTAT, is on the opposing strand. As a result, the orientation of the Pit-1 POU-specific domain relative to the POU-homeodomain is flipped, as compared to the Oct-1 crystal structure, indicating the remarkable flexibility of the POU-specific domain in adapting to variations in sequence within the site. Also in contrast to the Oct-1 monomer structure is the observation that the POU-specific and POU-homeodomain of each Pit-1 molecule make major groove contacts on the same face of the DNA, consistent with the constraints imposed by its 15 amino acid linker. As a result, the Pit-1 POU domain homodimer essentially surrounds its DNA binding site. In the Pit-1 POU domain homodimer the dimerization interface is formed between the C-terminal end of helix 3 of the POU-homeodomain of one Pit-1 molecule and the N-terminus of helix 1 and the loop between helices 3 and 4 of the POU-specific domain of the other Pit-1 molecule. In contrast to other homeodomain crystal structures, the C-terminus of helix 3 in the Pit-1 POU-homeo-domain has an extended structure. (ABSTRACT TRUNCATED)

Tumor suppressor Smad4 is a transforming growth factor beta-inducible DNA binding protein.

Members of the Smad family of proteins are thought to play important roles in transforming growth factor beta (TGF-beta)-mediated signal transduction. In response to TGF-beta, specific Smads become inducibly phosphorylated, form heteromers with Smad4, and undergo nuclear accumulation. In addition, overexpression of specific Smad combinations can mimic the transcriptional effect of TGF-beta on both the plasminogen activator inhibitor 1 (PAI-1) promoter and the reporter construct p3TP-Lux. Although these data suggest a role for Smads in regulating transcription, the precise nuclear function of these heteromeric Smad complexes remains largely unknown. Here we show that in Mv1Lu cells Smad3 and Smad4 form a TGF-beta-induced, phosphorylation-dependent, DNA binding complex that specifically recognizes a bipartite binding site within p3TP-Lux. Furthermore, we demonstrate that Smad4 itself is a DNA binding protein which recognizes the same sequence. Interestingly, mutations which eliminate the Smad DNA binding site do not interfere with either TGF-beta-dependent transcriptional activation or activation by Smad3/Smad4 cooverexpression. In contrast, mutation of adjacent AP1 sites within this context eliminates both TGF-beta-dependent transcriptional activation and activation in response to Smad3/Smad4 cooverexpression. Furthermore, concatemerized AP1 sites, in isolation, are activated by Smad3/Smad4 cooverexpression and, to a certain extent, by TGF-beta. Taken together, these data suggest that the Smad3/Smad4 complex has at least two separable nuclear functions: it forms a rapid, yet transient sequence-specific DNA binding complex, and it potentiates AP1-dependent transcriptional activation.

Identification of the fliI and fliJ components of the Caulobacter flagellar type III protein secretion system.

Caulobacter crescentus is motile by virtue of a polar flagellum assembled during the predivisional stage of the cell cycle. Three mutant strains in which flagellar assembly was blocked at an early stage were isolated. The mutations in these strains mapped to an operon of two genes, fliI and fliJ, both of which are necessary for motility. fliI encodes a 50-kDa polypeptide whose sequence is closely related to that of the Salmonella typhimurium FliI protein, an ATPase thought to energize the export of flagellar subunits across the cytoplasmic membrane through a type III protein secretion system. fliJ encodes a 16-kDa hydrophilic protein of unknown function. Epistasis experiments demonstrated that the fliIJ operon is located in class II of the C. crescentus flagellar regulatory hierarchy, suggesting that the gene products act at an early stage in flagellar assembly. The expression of fliIJ is induced midway through the cell cycle, coincident with other class II operons, but the FliI protein remains present throughout the cell cycle. Subcellular fractionation showed that FliI is present both in the cytoplasm and in association with the membrane. Mutational analysis of FliI showed that two highly conserved amino acid residues in a bipartite ATP binding motif are necessary for flagellar assembly.

Purification and characterization of FBI-1, a cellular factor that binds to the human immunodeficiency virus type 1 inducer of short transcripts.

The human immunodeficiency virus (HIV-1) promoter directs the synthesis of two classes of RNA molecules, short transcripts and full-length transcripts. The synthesis of short transcripts depends on a bipartite DNA element, the inducer of short transcripts (IST), located in large part downstream of the HIV-1 start site of transcription. IST does not require any viral product for function and is thought to direct the assembly of transcription complexes that are incapable of efficient elongation. Nothing is known, however, about the biochemical mechanisms that mediate IST function. Here, we report the identification and purification of a factor that binds specifically to the IST. This factor, FBI-1, recognizes a large bipartite binding site that coincides with the bipartite IST element. It is constituted at least in part by an 86-kDa polypeptide that can be specifically cross-linked to IST. FBI-1 also binds to promoter and attenuation regions of a number of cellular and viral transcription units that are regulated by a transcription elongation block. This observation, together with the observation that the binding of FBI-1 to IST mutants correlates with the ability of these mutants to direct IST function, suggests that FBI-1 may be involved in the establishment of abortive transcription complexes.

Maize Activator transposase has a bipartite DNA binding domain that recognizes subterminal sequences and the terminal inverted repeats

The mobility of maize transposable element Activator (Ac) is dependent on the 11-bp terminal inverted repeats (IRs) and approximately 250 subterminal nucleotides at each end. These sequences flank the coding region for the transposase (TPase) protein, which is required for the transposition reaction. Here we show that Ac TPase has a bipartite DNA binding domain, and recognizes the IRs and subterminal sequences in the Ac ends. TPase binds cooperatively to repetitive ACG and TCG sequences, of which 25 copies are found in the 5' and 20 copies in the 3' subterminal regions. TPase affinity is highest when these sites are flanked on the 3' side by an additional G residue (A/TCGG), which is found at 75% of binding sites. Moreover, TPase binds specifically to the Ac IRs, albeit with much lower affinity. Two mutations within the IRs that immobilize Ac abolish TPase binding completely. The basic DNA binding domain of TPase is split into two subdomains. Binding to the subterminal motifs is accomplished by the C-terminal subdomain alone, whereas recognition of the IRs requires the N-terminal subdomain in addition. Furthermore, TPase is extremely flexible in DNA binding. Two direct or inverted binding sites are bound equally well, and sites that are five to twelve bases apart are similarly well bound. The consequences of these findings for the Ac transposition reaction are discussed.

Molecular cloning and analysis of Schizosaccharomyces pombe Reb1p: sequence-specific recognition of two sites in the far upstream rDNA intergenic spacer.

The coding sequences for a Schizosaccharomyces pombe sequence-specific DNA binding protein, Reb1p, have been cloned. The predicted S. pombe Reb1p is 24-29% identical to mouse TTF-1 (transcription termination factor-1) and Saccharomyces cerevisiae REB1 protein, both of which direct termination of RNA polymerase I catalyzed transcripts. The S.pombe Reb1 cDNA encodes a predicted polypeptide of 504 amino acids with a predicted molecular weight of 58.4 kDa. The S. pombe Reb1p is unusual in that the bipartite DNA binding motif identified originally in S.cerevisiae and Klyveromyces lactis REB1 proteins is uninterrupted and thus S.pombe Reb1p may contain the smallest natural REB1 homologous DNA binding domain. Its genomic coding sequences were shown to be interrupted by two introns. A recombinant histidine-tagged Reb1 protein bearing the rDNA binding domain has two homologous, sequence-specific binding sites in the S. pomber DNA intergenic spacer, located between 289 and 480 nt downstream of the end of the approximately 25S rRNA coding sequences. Each binding site is 13-14 bp downstream of two of the three proposed in vivo termination sites. The core of this 17 bp site, AGGTAAGGGTAATGCAC, is specifically protected by Reb1p in footprinting analysis.

Cooperative interactions between paired domain and homeodomain.

The Pax proteins are a family of transcriptional regulators involved in many developmental processes in all higher eukaryotes. They are characterized by the presence of a paired domain (PD), a bipartite DNA binding domain composed of two helix-turn-helix (HTH) motifs,the PAI and RED domains. The PD is also often associated with a homeodomain (HD) which is itself able to form homo- and hetero-dimers on DNA. Many of these proteins therefore contain three HTH motifs each able to recognize DNA. However, all PDs recognize highly related DNA sequences, and most HDs also recognize almost identical sites. We show here that different Pax proteins use multiple combinations of their HTHs to recognize several types of target sites. For instance, the Drosophila Paired protein can bind, in vitro, exclusively through its PAI domain, or through a dimer of its HD, or through cooperative interaction between PAI domain and HD. However, prd function in vivo requires the synergistic action of both the PAI domain and the HD. Pax proteins with only a PD appear to require both PAI and RED domains, while a Pax-6 isoform and a new Pax protein, Lune, may rely on the RED domain and HD. We propose a model by which Pax proteins recognize different target genes in vivo through various combinations of their DNA binding domains, thus expanding their recognition repertoire.

Crystal structures of the trimeric human immunodeficiency virus type 1 matrix protein: implications for membrane association and assembly.

The human immunodeficiency virus type 1 (HIV-1) matrix protein forms a structural shell associated with the inner viral membrane and performs other essential functions throughout the viral life cycle. The crystal structure of the HIV-1 matrix protein, determined at 2.3 angstrom resolution, reveals that individual matrix molecules are composed of five major helices capped by a three-stranded mixed beta-sheet. Unexpectedly, the protein assembles into a trimer in three different crystal lattices, burying 1880 angstrom2 of accessible surface area at the trimer interfaces. Trimerization appears to create a large, bipartite membrane binding surface in which exposed basic residues could cooperate with the N-terminal myristoyl groups to anchor the protein on the acidic inner membrane of the virus.

Identification of Novel DNA Binding Sites Recognized by the Transcription Factor mPOU (POU6F1)

Transcription factors of the POU family recognize DNA through their POU domain which represents a bipartite DNA binding motif consisting of a POU specific domain and a POU homeobox. It is thought that both subdomains make specific contacts with DNA and participate in DNA binding. Here we identify novel DNA binding sites for the POU protein mPOU (POU6F1). The sites contain two TAAT motifs either as palindromes or as direct repeats. DNA binding is mediated through the POU homeobox only. Transactivation experiments revealed that mPOU per se showed no transcriptional activation but could act as a repressor of Oct2A mediated activation.

In Vivo Mutational Analysis of the DNA Binding Domain of the Tissue-specific Transcription Factor, Pit-1

Pit-1 is a member of the POU family of transcription factors, which contain a bipartite DNA binding domain. The DNA binding domain consists of a POU-specific domain and a POU homeodomain. Each of the subdomains can interact with DNA independently, but both subdomains are required for high affinity, sequence-specific DNA binding. To examine the contributions of individual amino acids to the function of the DNA binding domain of Pit-1, we developed an approach involving random, in vitro mutagenesis followed by functional screening in Saccharomyces cerevisiae. Using this strategy, we identified a number of point mutations that altered the function of the Pit-1 DNA binding domain. Mutations that altered Pit-1 function were found in both the POU-specific and the POU homeodomain. Most of the mutations involve amino acid residues that are conserved in POU factors. One of the more frequent kinds of mutation affected residues located in the hydrophobic core of the protein. Another common mutation involved amino acids that are thought to make specific contacts with DNA. These mutations define a number of amino acid residues that are important for the function of the DNA binding domain of Pit-1.

The origin recognition complex interacts with a bipartite DNA binding site within yeast replicators.

Replicators are genetically defined elements within chromosomes that determine the location of origins of DNA replication. In the yeast Saccharomyces cerevisiae, the ARS1 replicator contains multiple functional DNA elements: an essential A element and three important B elements--B1, B2, and B3. Functionally similar A, B1, and B2 elements are also present in the ARS307 replicator. The B3 element binds a replication and transcription enhancer protein Abf1p, whereas the A element is required for binding the origin recognition complex (ORC). The function of the B1 and B2 elements remains to be defined. We have used a gel-based DNA binding assay to study the interaction between replicators and the putative initiator protein ORC. In addition to the established requirements for ATP and the A element for ORC-DNA interaction, the new data demonstrate that sequences in the B1 element are also important for ORC-DNA association. This conclusion is supported by DNase I footprint analyses and demonstrates that ORC binds to a bipartitite recognition element within the DNA. Furthermore, mutation of nucleotides in the B1 element suggests that this element has other functions in the initiation of DNA replication besides participating in the ORC-DNA interaction.

Tc1 transposase of Caenorhabditis elegans is an endonuclease with a bipartite DNA binding domain.

The Tc1 transposon of Caenorhabditis elegans is a member of the Tc1/mariner family of mobile elements. These elements have inverted terminal repeats that flank a single transposase gene. Here we show that Tc1 transposase, Tc1A, has a bipartite DNA binding domain related to the paired domain of mammalian and Drosophila genes. Both the DNA binding domain of Tc1A and the DNA binding site in the inverted repeat of Tc1 can be divided into two subdomains. Methylation interference studies demonstrate adjacent minor and major groove contacts at the inner part of the binding site by the N-terminal 68 amino acids of the DNA binding domain. In addition, Tc1A amino acids 69-142 are essential for major groove contacts at the outer part of the binding site. Recombinant Tc1A is found to be able to introduce a single strand nick at the 5' end of the transposon in vitro. Furthermore, Tc1A can mediate a phosphoryl transfer reaction. A mutation in a DDE motif abolishes both endonucleolytic and phosphoryl transfer activities, suggesting that Tc1A carries a catalytic core common to retroviral integrases and IS transposases.

Mutational and Structural Analysis of the RNA Binding Site for Escherichia coli Ribosomal Protein S7

Ribosomal protein S7 binds to a small RNA fragment of about 100 nucleotides within the lower half of the 3′ major domain of E. coli 16 S rRNA. This fragment (D3M) comprises two large internal loops, A and B, connected by helix 29, a six-base-pair helix containing a G·U pair. Two hairpins with non-canonical base-pairs, 42′ and 43, protrude from loops A and B, respectively. We used site-directed mutagenesis and molecular probing to further define which parts of D3M are important for S7 binding. Changing the stem of hairpin 42′ into a Watson-Crick helix did not affect S7 binding, indicating that the non-canonical pairs of 42′ do not provide recognition features for S7. However, deletion of this hairpin decreased S7 binding affinity by about threefold and altered the conformation of loop A. Deletion of the upper part of hairpin 43 (the loop and the adjacent four base-pairs) did not affect S7 binding, whereas the lower part of this hairpin (three base-pairs) was to found to be required for proper S7 binding. Moreover, replacing the U·G pair with a C·G pair in this lower part decreased S7 binding affinity by twofold, suggesting that the U·G pair is a recognition signal for S7, S7 binding was also affected by mutations in helix 29. Insertion of the nucleotide 5′ to the G or 3′ to the U of the G·U pair decreased S7 binding affinity by about threefold and twofold, respectively, whereas replacement of the G·U pair by a G·C pair enhanced the affinity about twofold, and lengthening the helix by inserting a C·G pair upstream from the G·U pair had no effect. Taken together, these results are consistent with a bipartite binding site for S7 on 16 S rRNA, involving two regions of interaction: one centered around helix 29 and extending on the adjacent part of loop A, and the other one centered around the lower part of hairpin 43 and probably extending on the adjoining of loop B.

An element of the elastase I enhancer is an overlapping bipartite binding site activated by a heteromeric factor.

The B element of the elastase I transcriptional enhancer is active in both exocrine and endocrine cells of the pancreas. Cell transfection experiments revealed that in an acinar cell line the active sequence of the element is more extensive than in an endocrine cell line. Electrophoretic mobility shift assays identified three major complexes (designated C, M, and L) from acinar cell nuclear extracts bound to the element. The C complex appears to be responsible for the activity of the element in acinar cells because its binding site, determined by methylation interference and mobility shift competition experiments, matches the critical sequence identified by cell transfection analysis of mutated B elements. The DNA sequence requirements for formation of the C complex is the sum of those for the M and L complexes. Methylation interference experiments indicated that the sensitivity of the C complex to guanine methylation also was the sum of that of the M and L complexes. Diagonal electrophoretic mobility shift assays confirmed that L is a component of complex C. However, the M complex, which we identified as GATA-4, is not part of the C complex, because the C complex was neither competed by GATA-binding sites nor super-shifted by anti-GATA-4 antiserum. Both the C and L complexes are specific to the pancreatic acinar cell line.

Characterization of intra- and intermolecular DNA ligation mediated by eukaryotic topoisomerase I. Role of bipartite DNA interaction in the ligation process.

The capacity of eukaryotic topoisomerase I to catalyze intra- and intermolecular DNA strand transfer via a two-step cleavage/ligation reaction was investigated by use of purified enzyme and defined DNA substrates. Topoisomerase I-mediated cleavage requires separate interaction with a duplex region encompassing the cleavage site (region A) and a duplex region located on the side holding the 5'-OH end generated by cleavage (region B). Cleaved topoisomerase I-DNA complexes containing enzyme covalently attached at internal and terminal positions were employed to characterize the intra- and intermolecular ligation reactions. Enzyme attached covalently at an internal position of a partially single-stranded DNA molecule is able to catalyze ligation of a complementary dinucleotide within region A in the absence of interaction with region B. Moreover, the dinucleotide confines the minimal DNA acceptor for intramolecular ligation. Topoisomerase I attached covalently to DNA at a terminal position can ligate the cleaved strand to heterologous duplex DNA regardless of sequence, whereas ligation does not proceed with single-stranded DNA. When these features are considered together with the observation that intermolecular ligation is inhibited by 1 M NaCl, it suggests that the reaction requires bipartite DNA interaction. A model is proposed that relates the bipartite DNA binding of eukaryotic topoisomerase I to the catalytic functions.