Rous sarcoma virus integrase protein: mapping functions for catalysis and substrate binding.

Rous sarcoma virus (RSV), like all retroviruses, encodes an integrase protein that is responsible for covalently joining the reverse-transcribed viral DNA to host DNA. We have probed the organization of functions within RSV integrase by constructing mutant derivatives and assaying their activities in vitro. We find that deletion derivatives lacking the amino-terminal 53 amino acids, which contain the conserved H-X(3-7)-H-X(23-32)-C-X(2)-C (HHCC) Zn(2+)-binding motif, are greatly impaired in their ability to carry out two reactions characteristic of integrase proteins: specific cleavage of the viral DNA termini and DNA strand transfer. Deletion mutants lacking the carboxyl-terminal 69 amino acids are also unable to carry out these reactions. However, all deletion mutants that retain the central domain are capable of carrying out disintegration, an in vitro reversal of the normal DNA strand transfer reaction, indicating that the catalytic center probably lies within this central region. Another conserved motif, D-X(39-58)-D-X(35)-E, is found in this central domain. These findings with RSV integrase closely parallel previous findings with human immunodeficiency virus integrase, indicating that a modular catalytic domain is a general feature of this family of proteins. Surprisingly, and unlike results obtained so far with human immunodeficiency virus integrase, efficient strand transfer activity can be restored to a mutant RSV integrase lacking the amino-terminal HHCC domain by fusion to various short peptides. Furthermore, these fusion proteins retain the substrate specificity of RSV integrase. These data support a model in which the integrase activities required for strand transfer in vitro, including substrate recognition, multimerization, and catalysis, all lie primarily outside the amino-terminal HHCC domain.     

Characterization of human immunodeficiency virus type 1 integrase expressed in Escherichia coli and analysis of variants with amino-terminal mutations.

Replication of a retroviral genome depends upon integration of the viral DNA into a chromosome of the host cell. The integration reaction is mediated by integrase, a viral enzyme. Human immunodeficiency virus type 1 integrase was expressed in Escherichia coli and purified to near homogeneity. Optimum conditions for the integration and 3'-end-processing activities of integrase were characterized by using an in vitro assay with short, double-stranded oligonucleotide substrates. Mutants containing amino acid substitutions within the HHCC region, defined by phylogenetically conserved pairs of histidine and cysteine residues near the N terminus, were constructed and characterized by using three assays: 3'-end processing, integration, and the reverse of the integration reaction (or disintegration). Mutations in the conserved histidine and cysteine residues abolished both integration and processing activities. Weak activity in both assays was retained by two other mutants containing substitutions for less highly conserved amino acids in this region. All mutants retained activity in the disintegration assay, implying that the active site for DNA cleavage-ligation is not located in this domain and that the HHCC region is not the sole DNA-binding domain in the protein. However, the preferential impairment of processing and integration rather than disintegration by mutations in the HHCC region is consistent with a role for this domain in recognizing features of the viral DNA. This hypothesis is supported by the results of disintegration assays performed with altered substrates. The results support a model involving separate viral and target DNA-binding sites on integrase.     

Identification of the catalytic and DNA-binding region of the human immunodeficiency virus type I integrase protein.

The integrase (IN) protein of the human immunodeficiency virus (HIV) is required for specific cleavage of the viral DNA termini, and subsequent integration of the viral DNA into target DNA. To identify the various domains of the IN protein we generated a series of IN deletion mutants as fusions to maltose-binding protein (MBP). The deletion mutants were tested for their ability to bind DNA, to mediate site-specific cleavage of the viral DNA ends, and to carry out integration and disintegration reactions. We found that the DNA-binding region resides between amino acids 200 and 270 of the 288-residues HIV-1 IN protein. The catalytic domain of the protein was mapped between amino acids 50 and 194. For the specific activities of IN, cleavage of the viral DNA and integration, both the DNA-binding domain and the conserved amino-terminal region of IN are required. These regions are dispensable however, for disintegration activity.     

Identification of a subdomain of CENP-B that is necessary and sufficient for localization to the human centromere

We have combined in vivo and in vitro approaches to investigate the function of CENP-B, a major protein of human centromeric heterochromatin. Expression of epitope-tagged deletion derivatives of CENP-B in HeLa cells revealed that a single domain less than 158 residues from the amino terminus of the protein is sufficient to localize CENP-B to centromeres. Centromere localization was abolished if as few as 28 amino acids were removed from the amino terminus of CENP-B. The centromere localization signal of CENP-B can function in an autonomous fashion, relocating a fused bacterial enzyme to centromeres. The centromere localization domain of CENP-B specifically binds in vitro to a subset of alpha-satellite DNA monomers. These results suggest that the primary mechanism for localization of CENP-B to centromeres involves the recognition of a DNA sequence found at centromeres. Analysis of the distribution of this sequence in alpha-satellite DNA suggests that CENP-B binding may have profound effects on chromatin structure at centromeres.     

Localization of the active site of the alpha subunit of the Escherichia coli DNA polymerase III holoenzyme.

Using a deletion approach on the alpha subunit of DNA polymerase III from Escherichia coli, we show that there is an N-proximal polymerase domain which is distinct from a more C-proximal tau and beta binding domain. Although deletion of 60 residues from the alpha N terminus abolishes polymerase activity, deletions of 48, 169, and 342 amino acids from the C terminus progressively impair its catalytic efficiency but preserve an active site. Deletion of 342 C-terminal residues reduces k(cat) 46-fold, increases the Km for gapped DNA 5.5-fold, and increases the Km for deoxynucleoside triphosphates (dNTPs) twofold. The 818-residue protein with polymerase activity displays typical Michaelis-Menten behavior, catalyzing a polymerase reaction that is saturable with substrate and linear with time. With the aid of newly acquired sequences of the polymerase III alpha subunit from a variety of organisms, candidates for two key aspartate residues in the active site are identified at amino acids 401 and 403 of the E. coli sequence by inspection of conserved acidic amino acids. The motif Pro-Asp-X-Asp, where X is a hydrophobic amino acid, is shown to be conserved among all known DnaE proteins, including those from Bacillaceae, cyanobacteria, Mycoplasma, and mycobacteria. The E. coli DnaE deletion protein with only the N-terminal 366 amino acids does not have polymerase activity, consistent with the proposed position of the active-site residues.     

Activities of the feline immunodeficiency virus integrase protein produced in Escherichia coli.

Retroviral DNA integration requires the activity of at least one viral protein, the integrase (IN) protein. We cloned and expressed the integrase gene of feline immunodeficiency virus (FIV) in Escherichia coli as a fusion to the malE gene and purified the IN fusion protein by affinity chromatography. The protein is active in site-specific cleavage of the viral DNA ends, DNA strand transfer, and disintegration. FIV IN has a relaxed viral DNA substrate requirement: it cleaves and integrates FIV DNA termini, human immunodeficiency virus DNA ends, and Moloney murine leukemia virus DNA ends with high efficiencies. In the cleavage reaction, IN exposes a specific phosphodiester bond near the viral DNA end to nucleophilic attack. In vitro, either H2O, glycerol, or the 3' OH group of the viral DNA terminus can serve as nucleophile in this reaction. We found that FIV IN preferentially uses the 3' OH ends of the viral DNA as nucleophile, whereas HIV IN protein preferentially uses H2O and glycerol as nucleophiles.     

Substrate features important for recognition and catalysis by human immunodeficiency virus type 1 integrase identified by using novel DNA substrates.

The integrase encoded by human immunodeficiency virus type 1 (HIV-1) is required for integration of viral DNA into the host cell chromosome. In vitro, integrase mediates a concerted cleavage-ligation reaction (strand transfer) that results in covalent attachment of viral DNA to target DNA. With a substrate that mimics the strand transfer product, integrase carries out disintegration, the reverse of the strand transfer reaction, resolving this integration intermediate into its viral and target DNA parts. We used a set of disintegration substrates to study the catalytic mechanism of HIV-1 integrase and the interaction between the protein and the viral and target DNA sequence. One substrate termed dumbbell consists of a single oligonucleotide that can fold to form a structure that mimics the integration intermediate. Kinetic analysis using the dumbbell substrate showed that integrase turned over, establishing that HIV-1 integrase is an enzyme. Analysis of the disintegration activity on the dumbbell substrate and its derivatives showed that both the viral and target DNA parts of the molecule were required for integrase recognition. Integrase recognized target DNA asymmetrically: the target DNA upstream of the viral DNA joining site played a much more important role than the downstream target DNA in protein-DNA interaction. The site of transesterification was determined by both the DNA sequence of the viral DNA end and the structure of the branched substrate. Using a series of disintegration substrates with various base modifications, we found that integrase had relaxed structural specificity for the hydroxyl group used in transesterification and could tolerate distortion of the double-helical structure of these DNA substrates.     

Taq DNA聚合酶功能区域的定位

通过参Ｕ法定点突变产生了ＴａｑＤＮＡ聚合酶Ｎ端分别缺失３个,２３５个,２８７个和４４３个氨基酸的４个缺失体,利用Ｂａｌ－３１连续缺失法产生了ＴａｑＤＮＡ聚合酶的Ｃ端分别缺失了２个、１６个、２９个、３２个、３４个氨基酸的５个缺失体．经ＤＮＡ聚合酶活性测定表明Ｎ端缺失３个,２３５个,２８７个氨基酸后活力和完整的Ｔａｑ相近,而缺失４４３个氨基酸后则失去了ＤＮＡ聚合酶活力;Ｃ端的５个缺失体都失去了ＤＮＡ聚合酶活性．据此ＴａｑＤＮＡ聚合酶的功能区域被定位在２８７～８３２氨基酸之间．     

Intermolecular disintegration and intramolecular strand transfer activities of wild-type and mutant HIV-1 integrase.

We report the activities of HIV integrase protein on a novel DNA substrate, consisting of a pair of gapped duplex molecules. Integrase catalyzed an intermolecular disintegration reaction that requires positioning of a pair of the gapped duplexes in a configuration that resembles the intgration intermediate. However, the major reaction resulted from an intramolecular reaction involving a single gapped duplex, giving rise to a hairpin. Surprisingly, a deletion mutant of integrase that lacks both the amino and carboxyl terminal regions still catalyzed the intermolecular disintegration reaction, but supported only a very low level of the intramolecular reaction. The central core region of integrase is therefore sufficient to both bind the gapped duplex DNA and juxtapose a pair of such molecules through protein-protein interactions. We suggest that the branched DNA structures of the previously reported disintegration substrate, and the intermolecular disintegration substrate described here, assist in stabilizing protein-protein interactions that otherwise require the amino and carboxy terminal regions of integrase.     

Mutations in DNA Binding and Transactivation Domains Affect the Dynamics of Parvovirus NS1 Protein

The multifunctional replication protein of autonomous parvoviruses, NS1, is vital for viral genome replication and for the control of viral protein production. Two DNA-interacting domains of NS1, the N-terminal and helicase domains, are necessary for these functions. In addition, the N and C termini of NS1 are required for activation of viral promoter P38. By comparison with the structural and biochemical data from other parvoviruses, we identified potential DNA-interacting amino acid residues from canine parvovirus NS1. The role of the identified amino acids in NS1 binding dynamics was studied by mutagenesis, fluorescence recovery after photobleaching, and computer simulations. Mutations in the predicted DNA-interacting amino acids of the N-terminal and helicase domains increased the intranuclear binding dynamics of NS1 dramatically. A substantial increase in binding dynamics was also observed for NS1 mutants that targeted the metal ion coordination site in the N terminus. Interestingly, contrary to other mutants, deletion of the C terminus resulted in slower binding dynamics of NS1. P38 transactivation was severely reduced in both N-terminal DNA recognition and in C-terminal deletion mutants. These data suggest that the intranuclear dynamics of NS1 are largely characterized by its sequence-specific and -nonspecific binding to double-stranded DNA. Moreover, binding of NS1 is equally dependent on the N-terminal domain and conserved β-loop of the helicase domain.     

Genetic analysis of the human immunodeficiency virus type 1 integrase protein.

Single-amino-acid changes in a highly conserved central region of the human immunodeficiency virus type 1 (HIV-1) integrase protein were analyzed for their effects on viral protein synthesis, virion morphogenesis, and viral replication. Alteration of two amino acids that are invariant among retroviral integrases, D116 and E152 of HIV-1, as well as a mutation of the highly conserved amino acid S147 blocked viral replication in two CD4+ human T-cell lines. Mutations of four other highly conserved amino acids in the region had no detectable effect on viral replication, whereas mutations at two positions, N117 and Y143, resulted in viruses with a delayed-replication phenotype. Defects in virion precursor polypeptide processing, virion morphology, or viral DNA synthesis were observed for all of the replication-defective mutants, indicating that changes in integrase can have pleiotropic effects on viral replication.     

The DNMT1 target recognition domain resides in the N terminus

DNA-cytosine-5-methyltransferase 1 (DNMT1) is the enzyme believed to be responsible for maintaining the epigenetic information encoded by DNA methylation patterns. The target recognition domain of DNMT1, the domain responsible for recognizing hemimethylated CGs, is unknown. However, based on homology with bacterial cytosine DNA methyltransferases it has been postulated that the entire catalytic domain, including the target recognition domain, is localized to 500 amino acids at the C terminus of the protein. The N-terminal domain has been postulated to have a regulatory role, and it has been suggested that the mammalian DNMT1 is a fusion of a prokaryotic methyltransferase and a mammalian DNA-binding protein. Using a combination of in vitro translation of different DNMT1 deletion mutant peptides and a solid-state hemimethylated substrate, we show that the target recognition domain of DNMT1 resides in the N terminus (amino acids 122-417) in proximity to the proliferating cell nuclear antigen binding site. Hemimethylated CGs were not recognized specifically by the postulated catalytic domain. We have previously shown that the hemimethylated substrates utilized here act as DNMT1 antagonists and inhibit DNA replication. Our results now indicate that the DNMT1-PCNA interaction can be disrupted by substrate binding to the DNMT1 N terminus. These results point toward new directions in our understanding of the structure-function of DNMT1.     

Irreversible inhibition of human immunodeficiency virus type 1 integrase by dicaffeoylquinic acids

Human immunodeficiency virus type 1 (HIV-1) and other retroviruses require integration of a double-stranded DNA copy of the RNA genome into the host cell chromosome for productive infection. The viral enzyme, integrase, catalyzes the integration of retroviral DNA and represents an attractive target for developing antiretroviral agents. We identified several derivatives of dicaffeoylquinic acids (DCQAs) that inhibit HIV-1 replication in tissue culture and catalytic activities of HIV-1 integrase in vitro. The specific step at which DCQAs inhibit the integration in vitro and the mechanism of inhibition were examined in the present study. Titration experiments with different concentrations of HIV-1 integrase or DNA substrate found that the effect of DCQAs was exerted on the enzyme and not the DNA. In addition to HIV-1, DCQAs also inhibited the in vitro activities of MLV integrase and truncated variants of feline immunodeficiency virus integrase, suggesting that these compounds interacted with the central core domain of integrase. The inhibition on retroviral integrases was relatively specific, and DCQAs had no effect on several other DNA-modifying enzymes and phosphoryltransferases. Kinetic analysis and dialysis experiments showed that the inhibition of integrase by DCQAs was irreversible. The inhibition did not require the presence of a divalent cation and was unaffected by preassembling integrase onto viral DNA. The results suggest that the irreversible inhibition by DCQAs on integrase is directed toward conserved amino acid residues in the central core domain during catalysis.     

Human immunodeficiency virus type 1 integrase: effects of mutations on viral ability to integrate, direct viral gene expression from unintegrated viral DNA templates, and sustain viral propagation in primary cells.

Integrase is the only viral protein necessary for integration of retroviral DNA into chromosomal DNA of the host cell. Biochemical analysis of human immunodeficiency virus type 1 (HIV-1) integrase with purified protein and synthetic DNA substrates has revealed extensive information regarding the mechanism of action of the enzyme, as well as identification of critical residues and functional domains. Since in vitro reactions are carried out in the absence of other viral proteins and they analyze strand transfer of only one end of the donor substrate, they do not define completely the process of integration as it occurs during the course of viral infection. In an effort to further understand the role of integrase during viral infection, we initially constructed a panel of 24 HIV-1 mutants with specific alanine substitutions throughout the integrase coding region and analyzed them in a human T-cell line infection. Of these mutant viruses, 12 were capable of sustained viral replication, 11 were replication defective, and 1 was temperature sensitive for viral growth. The replication defective viruses express and correctly process the integrase and Gag proteins. Using this panel of mutants and an additional set of 18 mutant viruses, we identified nine amino acids which, when replaced with alanine, destroy integrase activity. Although none of the replication-defective mutants are able to integrate into the host genome, a subset of them with alterations in the catalytic triad are capable of Tat-mediated transactivation of an indicator gene linked to the viral long terminal repeat promoter. We present evidence that integration of the HIV-1 provirus is essential not only for productive infection of T cells but also for virus passage in both cultured peripheral blood lymphocytes and macrophage cells.     

A mutation at one end of Moloney murine leukemia virus DNA blocks cleavage of both ends by the viral integrase in vivo.

The integration of retroviral DNA proceeds through two steps: trimming of the termini to expose new 3' OH ends, and the transfer of those ends to the phosphates of target DNA. We have examined the ability of the Moloney murine leukemia virus integrase protein (IN) to trim the termini of the preintegrative DNA of mutant viruses with alterations in the U3 inverted repeat. The mutant terminus of one replication-defective viral DNA, containing a 7-bp deletion in the U3 inverted repeat, was not trimmed to produce the normal recessed end. Remarkably, the other terminus of this mutant DNA was also not trimmed, even though its sequence is wild type. This finding suggests that the IN protein requires the presence of two good ends before becoming properly activated to trim either one.     

Characterization of the minimal DNA-binding domain of the HIV integrase protein.

The human immunodeficiency virus (HIV) integrase (IN) protein mediates an essential step in the retroviral lifecycle, the integration of viral DNA into human DNA. A DNA-binding domain of HIV IN has previously been identified in the C-terminal part of the protein. We tested truncated proteins of the C-terminal region of HIV-1 IN for DNA binding activity in two different assays: UV-crosslinking and southwestern blot analysis. We found that a polypeptide fragment of 50 amino acids (IN220-270) is sufficient for DNA binding. In contrast to full-length IN protein, this domain is soluble under low salt conditions. DNA binding of IN220-270 to both viral DNA and non-specific DNA occurs in an ion-independent fashion. Point mutations were introduced in 10 different amino acid residues of the DNA-binding domain of HIV-2 IN. Mutation of basic amino acid K264 results in strong reduction of DNA binding and of integrase activity.     

Role of the UL25 protein in herpes simplex virus DNA encapsidation

The herpes simplex virus protein UL25 attaches to the external vertices of herpes simplex virus type 1 capsids and is required for the stable packaging of viral DNA. To define regions of the protein important for viral replication and capsid attachment, the 580-amino-acid UL25 open reading frame was disrupted by transposon mutagenesis. The UL25 mutants were assayed for complementation of a UL25 deletion virus, and in vitro-synthesized protein was tested for binding to UL25-deficient capsids. Of the 11 mutants analyzed, 4 did not complement growth of the UL25 deletion mutant, and analysis of these and additional mutants in the capsid-binding assay demonstrated that UL25 amino acids 1 to 50 were sufficient for capsid binding. Several UL25 mutations were transferred into recombinant viruses to analyze the effect of the mutations on UL25 capsid binding and on DNA cleavage and packaging. Studies of these mutants demonstrated that amino acids 1 to 50 of UL25 are essential for its stable interaction with capsids and that the C terminus is essential for DNA packaging and the production of infectious virus through its interactions with other viral packaging or tegument proteins. Analysis of viral DNA cleavage demonstrated that in the absence of a functional UL25 protein, aberrant cleavage takes place at the unique short end of the viral genome, resulting in truncated viral genomes that are not retained in capsids. Based on these observations, we propose a model where UL25 is required for the formation of DNA-containing capsids by acting to stabilize capsids that contain full-length viral genomes.     

Characterization of a DNA binding domain in the C-terminus of HIV-1 integrase by deletion mutagenesis.

The integrase (IN) protein of human immunodeficiency virus type 1 (HIV-1) catalyzes site-specific cleavage of 2 bases from the viral long terminal repeat (LTR) sequence yet it binds DNA with little DNA sequence specificity. We have previously demonstrated that the C-terminal half of IN (amino acids 154-288) possesses a DNA binding domain. In order to further characterize this region, a series of clones expressing truncated forms of IN as N-terminal fusion proteins in E.coli were constructed and analyzed by Southwestern blotting. Proteins containing amino acids 1-263, 1-248 and 170-288 retained the ability to bind DNA, whereas a protein containing amino acids 1-180 showed no detectable DNA binding. This defines a DNA binding domain contained within amino acids 180-248. This region contains an arrangement of 9 lysine and arginine residues each separated by 2-4 amino acids (KxxxKxxxKxxxxRxxxRxxRxxxxKxxxKxxxK), spanning amino acids 211-244, which is conserved in all HIV-1 isolates. A clone expressing full-length IN with a C-terminal fusion of 16 amino acids was able to bind DNA comparably to a cloned protein with a free C-terminus, and an IN-specific monoclonal antibody which recognizes an epitope contained within amino acids 264-279 was unable to block DNA binding, supporting the evidence that a region necessary for binding lies upstream of amino acid 264.