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1.
Biochemistry ; 40(38): 11596-603, 2001 Sep 25.
Article in English | MEDLINE | ID: mdl-11560510

ABSTRACT

The Escherichia coli Ada protein repairs methylphosphotriesters in DNA through direct, irreversible transfer to a cysteine residue on the protein, Cys 69. Methylation of Cys 69 increases the sequence-specific DNA-binding activity of Ada by 10(3)-fold, enabling the methylated protein to activate transcription of a methylation-resistance regulon. The thiolate sulfur atom of Cys 69 is coordinated to a tightly bound zinc ion in the Ada N-terminal domain, and this metal-ligand interaction plays a direct role in promoting the DNA repair chemistry. Ada is thus the founding member of a mechanistic class of proteins that employ metalloactivated thiolates as nucleophiles, other examples of which include protein prenyltransferases and cobalamin-independent methionine synthase. Here we have probed the role of the three other Cys residues in Ada that together with Cys 69 coordinate the zinc through mutation to the alternative ligand residues Asp and His. All of the mutant proteins folded properly and bound zinc, but none of them exhibited measurable levels of DNA repair activity. Significantly, the Cys-to-His mutant proteins retained nearly wild-type sequence-specific DNA-binding activity in the unmethylated state. These findings demonstrate that the three "spectator" Cys ligands communicate chemically with Cys 69 through the bound metal ion.


Subject(s)
Bacterial Proteins/metabolism , DNA Repair , DNA/metabolism , Escherichia coli Proteins , Escherichia coli/genetics , Escherichia coli/metabolism , Sulfhydryl Compounds/metabolism , Bacterial Proteins/chemistry , Base Sequence , Binding Sites , Cysteine , DNA/chemistry , Molecular Conformation , Nuclear Magnetic Resonance, Biomolecular , O(6)-Methylguanine-DNA Methyltransferase , Quantum Theory , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Substrate Specificity , Sulfhydryl Compounds/chemistry , Transcription Factors , Zinc/analysis
2.
EMBO J ; 20(15): 4278-86, 2001 Aug 01.
Article in English | MEDLINE | ID: mdl-11483530

ABSTRACT

Gene-targeted mice deficient in the evolutionarily conserved uracil-DNA glycosylase encoded by the UNG gene surprisingly lack the mutator phenotype characteristic of bacterial and yeast ung(-) mutants. A complementary uracil-DNA glycosylase activity detected in ung(-/-) murine cells and tissues may be responsible for the repair of deaminated cytosine residues in vivo. Here, specific neutralizing antibodies were used to identify the SMUG1 enzyme as the major uracil-DNA glycosylase in UNG-deficient mice. SMUG1 is present at similar levels in cell nuclei of non-proliferating and proliferating tissues, indicating a replication- independent role in DNA repair. The SMUG1 enzyme is found in vertebrates and insects, whereas it is absent in nematodes, plants and fungi. We propose a model in which SMUG1 has evolved in higher eukaryotes as an anti-mutator distinct from the UNG enzyme, the latter being largely localized to replication foci in mammalian cells to counteract de novo dUMP incorporation into DNA.


Subject(s)
Cytosine/metabolism , DNA Damage , DNA Glycosylases , DNA Repair , N-Glycosyl Hydrolases/metabolism , Nucleoside Deaminases/metabolism , Animals , Antibodies/immunology , Antibody Specificity , Base Pair Mismatch , Cell Division , Cytosine Deaminase , Evolution, Molecular , Genome , Guanine , Humans , Kinetics , Mice , Mice, Knockout , N-Glycosyl Hydrolases/genetics , N-Glycosyl Hydrolases/immunology , Sequence Homology, Amino Acid , Substrate Specificity , Uracil , Uracil-DNA Glycosidase , Vertebrates/genetics
3.
Org Lett ; 3(14): 2157-9, 2001 Jul 12.
Article in English | MEDLINE | ID: mdl-11440568

ABSTRACT

[reaction: see text] This report describes the parallel synthesis of all 16 stereoisomers of the cis-1,5 enediol module 1. Compounds 1 derive from 2 by silicon-tethered ring-closing metathesis. Such libraries of stereodiversified ligands provide a unique approach to ligand discovery that employs exhaustive searching of conformational space.


Subject(s)
Alcohols/chemical synthesis , Alkenes/chemical synthesis , Silanes/chemical synthesis , Alcohols/chemistry , Alkenes/chemistry , Cyclization , Fluorenes/chemical synthesis , Fluorenes/chemistry , Molecular Structure , Oligopeptides/chemistry , Silanes/chemistry , Stereoisomerism , Structure-Activity Relationship
5.
J Am Chem Soc ; 123(3): 398-408, 2001 Jan 24.
Article in English | MEDLINE | ID: mdl-11456541

ABSTRACT

Small molecules that induce or stabilize the association of macromolecules have proven to be useful effectors of a wide variety of biological processes. To date, all examples of such chemical inducers of dimerization have involved known ligands to well-characterized proteins. The generality of this approach could be broadened by enabling the discovery of heterodimerizers that target known macromolecules having no established ligand, or heterodimerizers that produce a novel biologic response in screens having no predetermined macromolecular target. Toward this end, we report the construction of a diversified library of synthetic heterodimerizers consisting of an invariant ligand that targets the FK506-binding protein (AP1867) attached to 320 substituted tetrahydrooxazepines (THOXs). The THOX components were generated by a combination of liquid- and solid-phase procedures employing sequential Mitsonobu displacements to join two structurally diversified olefin-containing monomers, followed by ruthenium-mediated olefin metathesis to effect closure of the seven-membered ring. The 320 resin-bound THOX ligands were coupled in parallel to AP1867, and the products were released from the resin to yield candidate heterodimerizers in sufficient yield and purity to be used directly in biologic testing. A representative panel of 25 candidate heterodimerizers were tested for their ability to pass through the membrane of human fibrosarcoma cells, and all were found to possess activity in this tissue culture system. These studies pave the way for further studies aimed at using small-molecule inducers of heterodimerization to effect novel biological responses in intact cells.


Subject(s)
Carrier Proteins/chemical synthesis , Cell Membrane Permeability , Combinatorial Chemistry Techniques , Biological Transport , Carrier Proteins/metabolism , Dimerization , Humans , Ligands , Protein Binding , Recombinant Fusion Proteins , Tacrolimus/analogs & derivatives , Tacrolimus/chemical synthesis , Tacrolimus/metabolism , Tacrolimus Binding Proteins/metabolism , Tumor Cells, Cultured
6.
Org Lett ; 3(1): 71-4, 2001 Jan 11.
Article in English | MEDLINE | ID: mdl-11429875

ABSTRACT

[figure: see text] We have developed a method for interference footprinting of contacted phosphates in protein-DNA complexes. Template-directed enzymatic polymerization using a synthetic triphosphate analogue (alpha Me-dTTP) generates a product having a modified Internucleotide linkage, which perturbs protein-phosphate contacts. We found that treatment of the methylphosphonodiester-substituted extension product under nonaqueous conditions (MeO-/MeOH) led to the formation of a single cleavage product at each T residue but to two cleavage products when treated under the standard aqueous piperidine cleavage protocol.


Subject(s)
DNA Footprinting/methods , DNA/chemistry , Peptide Mapping/methods , Proteins/chemistry , Catalysis , DNA/drug effects , Electrophoresis, Polyacrylamide Gel , Humans , Molecular Structure , Phosphates/chemistry , Proteins/drug effects , Structure-Activity Relationship
7.
Biochemistry ; 40(14): 4261-71, 2001 Apr 10.
Article in English | MEDLINE | ID: mdl-11284682

ABSTRACT

The Escherichia coli protein Ada specifically repairs the S(p) diastereomer of DNA methyl phosphotriesters in DNA by direct and irreversible transfer of the methyl group to its own Cys 69 which is part of a zinc-thiolate center. The methyl transfer converts Ada into a transcriptional activator that binds sequence-specifically to promoter regions of its own gene and other methylation resistance genes. Ada thus acts as a chemosensor to activate repair mechanisms in situations of methylation damage. Here we present a highly refined solution structure of the 10 kDa N-terminal domain, N-Ada10, which reveals structural details of the nonspecific DNA interaction of N-Ada10 during the repair process and provides a basis for understanding the mechanism of the conformational switch triggered by methyl transfer. To further elucidate this, EXAFS (extended X-ray absorption fine structure) and XANES (X-ray absorption near-edge structure) data were acquired, which confirmed that the zinc-thiolate center is maintained when N-Ada is methylated. Thus, ligand exchange is not the mechanism that enhances sequence-specific DNA binding and transcriptional activation upon methylation of N-Ada. The mechanism of the switch was further elucidated by recording NOESY spectra of specifically labeled methylated-Ada/DNA complexes, which showed that the transferred methyl group makes many contacts within N-Ada but none with the DNA. This implies that methylation of N-Ada induces a structural change, which enhances the promoter affinity of a remodeled surface region that does not include the transferred methyl group.


Subject(s)
Bacterial Proteins/chemistry , Escherichia coli Proteins , Escherichia coli/chemistry , Crystallography, X-Ray , DNA Methylation , DNA-Binding Proteins/chemistry , Ligands , Nuclear Magnetic Resonance, Biomolecular , O(6)-Methylguanine-DNA Methyltransferase , Organometallic Compounds/chemistry , Protein Conformation , Protein Structure, Secondary , Spectrum Analysis , Structure-Activity Relationship , Trans-Activators/chemistry , Transcription Factors , X-Rays , Zinc/chemistry
8.
Org Lett ; 2(25): 3999-4002, 2000 Dec 14.
Article in English | MEDLINE | ID: mdl-11112627

ABSTRACT

[structure] This report describes a modular approach to the synthesis of stereodiversified natural product-like libraries. Monomers 2 and 3 were coupled in parallel by silyl-tethered olefin metathesis to generate all 16 stereoisomers of cis-enediols 1. All 16 stereoisomers were incorporated into chimerae having flanking peptidic segments. These chimerae exhibited a broad range of hydrophobicities, raising the possibility that stereochemical variation might be used to tune the pharmacologic properties of small molecules.


Subject(s)
Combinatorial Chemistry Techniques , Ligands , Alkenes/chemical synthesis , Catalysis , Chromatography, High Pressure Liquid , Peptides/chemical synthesis , Siloxanes/chemical synthesis , Siloxanes/chemistry , Stereoisomerism
9.
J Biol Chem ; 275(47): 36506-8, 2000 Nov 24.
Article in English | MEDLINE | ID: mdl-11084051

ABSTRACT

Cytosine deamination and the misincorporation of 2'-dUrd into DNA during replication result in the presence of uracil in DNA. Uracil-DNA glycosylases (UDGs) initiate the excision repair of this aberrant base by catalyzing the hydrolysis of the N-glycosidic bond. UDGs are expressed by nearly all known organisms, including some viruses, in which the functional role of the UDG protein remains unresolved. This issue could in principle be addressed by the availability of designed synthetic inhibitors that target the viral UDG without affecting the endogenous human UDG. Here, we report that double-stranded and single-stranded oligonucleotides incorporating either of two dUrd analogs tightly bind and inhibit the activity of herpes simplex virus type-1 (HSV-1) UDG. Both inhibitors are exquisitely specific for the HSV-1 UDG over the human UDG. These inhibitors should prove useful in structural studies aimed at understanding substrate recognition and catalysis by UDGs, as well as in elucidating the biologic role of UDGs in the life cycle of herpesviruses.


Subject(s)
DNA Glycosylases , Enzyme Inhibitors/chemical synthesis , Floxuridine/chemistry , Furans/chemistry , Herpesvirus 1, Human/enzymology , N-Glycosyl Hydrolases/antagonists & inhibitors , Base Sequence , Electrophoresis, Polyacrylamide Gel , Enzyme Inhibitors/pharmacology , Humans , Isomerism , Models, Chemical , Molecular Mimicry , Molecular Sequence Data , Uracil-DNA Glycosidase
10.
Biochemistry ; 39(32): 9725-36, 2000 Aug 15.
Article in English | MEDLINE | ID: mdl-10933789

ABSTRACT

HMG-D is an abundant high mobility group chromosomal protein present during early embryogenesis in Drosophila melanogaster. It is a non-sequence-specific member of a protein family that uses the HMG domain for binding to DNA in the minor groove. The highly charged C-terminal tail of HMG-D contains AK motifs that contribute to high-affinity non-sequence-specific DNA binding. To understand the interactions of the HMG domain and C-terminal tail of HMG-D with DNA in solution, a complex between a high-affinity truncated form of the protein and a disulfide cross-linked DNA fragment was studied using heteronuclear NMR techniques. Despite its relatively high affinity for the single "prebent" site on the DNA, K(d) = 1.4 nM, HMG-D forms a non-sequence-specific complex with the DNA as indicated by exchange broadening of the protein resonances at the DNA interface in solution. The secondary structural elements of the protein are preserved when the protein is complexed with the DNA, and the DNA-binding interface maps to the regions of the protein where the largest chemical shift differences occur. The C-terminal tail of HMG-D confers high-affinity DNA binding, has an undefined structure, and appears to make direct contacts in the major groove of DNA via residues that are potentially regulated by phosphorylation. We conclude that while the HMG domain of HMG-D recognizes DNA with a mode of binding similar to that used by the sequence-specific HMG domain transcription factors, there are noteworthy differences in the structure and interactions of the C-terminal end of the DNA-binding domain and the C-terminal tail.


Subject(s)
DNA-Binding Proteins/chemistry , DNA/chemistry , High Mobility Group Proteins/chemistry , Insect Proteins/chemistry , Amino Acid Sequence , Animals , Binding Sites , Carbon Isotopes , DNA/metabolism , DNA-Binding Proteins/metabolism , Deuterium , Drosophila melanogaster , Insect Proteins/metabolism , Models, Molecular , Molecular Sequence Data , Nitrogen Isotopes , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Structure, Tertiary , Sequence Homology, Amino Acid
11.
Proc Natl Acad Sci U S A ; 97(13): 7130-5, 2000 Jun 20.
Article in English | MEDLINE | ID: mdl-10860980

ABSTRACT

NFATc (a member of the family of nuclear factors of activated T cells) is a transcriptional factor responsible for the Ca(2+)-inducible activation of cytokine genes during the immune response. In resting T cells, NFATc is retained in the cytoplasm by a mechanism that depends on multiple phosphorylations in an N-terminal regulatory domain. Physical interaction with and dephosphorylation by Ca(2+)-activated calcineurin (Cn) allows the protein to enter the nucleus, where it binds to specific sites in cytokine gene promoters. Previous studies had identified a peptide segment in NFATc that binds Cn stably. Here we report the identification of a second Cn-binding element in NFATc, which synergizes with the previously identified element. Although these sequences are conserved in all isoforms of NFAT, we find that the two sites contribute differentially to the overall affinity for Cn in an isoform-dependent manner. The regulatory domain of NFAT also was found to be entirely devoid of structure, both in the phosphorylated and unphosphorylated state. This finding suggests that the NFAT regulatory domain does not undergo phosphorylation-induced conformational switching, but instead requires partner proteins to control accessibility of the NFAT nuclear localization and nuclear export signals.


Subject(s)
Calcineurin/metabolism , DNA-Binding Proteins/genetics , Nuclear Proteins/genetics , Transcription Factors/genetics , Amino Acid Sequence , Binding Sites/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Humans , Molecular Sequence Data , NFATC Transcription Factors , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Protein Binding , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Transcription Factors/chemistry , Transcription Factors/metabolism
12.
Chem Biol ; 7(5): 355-64, 2000 May.
Article in English | MEDLINE | ID: mdl-10801473

ABSTRACT

BACKGROUND: HIV-1 reverse transcriptase (RT) is a major target for the treatment of acquired immunodeficiency syndrome (AIDS). Resistance mutations in RT compromise treatment, however. Efforts to understand the enzymatic mechanism of RT and the basis for mutational resistance to anti-RT drugs have been hampered by the failure to crystallize a catalytically informative RT-substrate complex. RESULTS: We present here experiments that allow us to understand the reason for the failure to crystallize such a complex. Based on this understanding, we have devised a new approach for using a combinatorial disulfide cross-linking strategy to trap a catalytic RT*template:primer*dNTP ternary complex, thereby enabling the growth of co-crystals suitable for high-resolution structural analysis. The crystals contain a fully assembled active site poised for catalysis. The cross-link itself appears to be conformationally mobile, and the surrounding region is undistorted, suggesting that the cross-link is a structurally passive device that merely acts to prevent dissociation of the catalytic complex. CONCLUSIONS: The new strategy discussed here has resulted in the crystallization and structure determination of a catalytically relevant RT*template:primer*dNTP complex. The structure has allowed us to analyze possible causes of drug resistance at the molecular level. This information will assist efforts to develop new classes of nucleoside analog inhibitors, which might help circumvent current resistance profiles. The covalent trapping strategy described here may be useful with other protein-DNA complexes that have been refractory to structural analysis.


Subject(s)
DNA Primers/chemistry , Disulfides/chemistry , HIV Reverse Transcriptase/chemistry , HIV Reverse Transcriptase/isolation & purification , Catalysis , Cross-Linking Reagents/chemistry , Crystallography, X-Ray , Electrophoresis, Polyacrylamide Gel , Genetic Vectors/chemical synthesis , HIV Reverse Transcriptase/biosynthesis , HIV Reverse Transcriptase/genetics , Nucleic Acid Heteroduplexes/chemistry , RNA/chemistry , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Sulfhydryl Compounds/chemistry , Templates, Genetic
13.
Nature ; 403(6772): 859-66, 2000 Feb 24.
Article in English | MEDLINE | ID: mdl-10706276

ABSTRACT

Spontaneous oxidation of guanine residues in DNA generates 8-oxoguanine (oxoG). By mispairing with adenine during replication, oxoG gives rise to a G x C --> T x A transversion, a frequent somatic mutation in human cancers. The dedicated repair pathway for oxoG centres on 8-oxoguanine DNA glycosylase (hOGG1), an enzyme that recognizes oxoG x C base pairs, catalysing expulsion of the oxoG and cleavage of the DNA backbone. Here we report the X-ray structure of the catalytic core of hOGG1 bound to oxoG x C-containing DNA at 2.1 A resolution. The structure reveals the mechanistic basis for the recognition and catalytic excision of DNA damage by hOGG1 and by other members of the enzyme superfamily to which it belongs. The structure also provides a rationale for the biochemical effects of inactivating mutations and polymorphisms in hOGG1. One known mutation, R154H, converts hOGG1 to a promutator by relaxing the specificity of the enzyme for the base opposite oxoG.


Subject(s)
DNA Repair , DNA/chemistry , Escherichia coli Proteins , Guanine/analogs & derivatives , Mutagens/chemistry , N-Glycosyl Hydrolases/chemistry , Amino Acid Sequence , Catalysis , Catalytic Domain , Cloning, Molecular , Crystallography, X-Ray , Cytosine/chemistry , DNA/metabolism , DNA-Formamidopyrimidine Glycosylase , Escherichia coli , Guanine/chemistry , Guanine/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Mutagens/metabolism , Mutation , Nucleic Acid Conformation , Protein Binding , Protein Conformation , Sequence Alignment , Structure-Activity Relationship
15.
Proc Natl Acad Sci U S A ; 96(26): 14801-6, 1999 Dec 21.
Article in English | MEDLINE | ID: mdl-10611293

ABSTRACT

Transcriptional activation domains share little sequence homology and generally lack folded structures in the absence of their targets, aspects that have rendered activation domains difficult to characterize. Here, a combination of biochemical and nuclear magnetic resonance experiments demonstrates that the activation domain of the tumor suppressor p53 has an FXXPhiPhi motif (F, Phe; X, any amino acids; Phi, hydrophobic residues) that folds into an alpha-helix upon binding to one of its targets, hTAF(II)31 (a human TFIID TATA box-binding protein-associated factor). MDM2, the cellular attenuator of p53, discriminates the FXXPhiPhi motif of p53 from those of NF-kappaB p65 and VP16 and specifically inhibits p53 activity. Our studies support the notion that the FXXPhiPhi sequence is a general alpha-helical recognition motif for hTAF(II)31 and provide insights into the mechanistic basis for regulation of p53 function.


Subject(s)
Nuclear Proteins , Proto-Oncogene Proteins/metabolism , TATA-Binding Protein Associated Factors , Trans-Activators/metabolism , Transcription Factor TFIID , Transcriptional Activation , Tumor Suppressor Protein p53/metabolism , Amino Acid Motifs , Amino Acid Sequence , Humans , Molecular Sequence Data , Protein Binding , Protein Structure, Secondary , Proto-Oncogene Proteins c-mdm2 , Tumor Suppressor Protein p53/chemistry
16.
EMBO J ; 18(23): 6599-609, 1999 Dec 01.
Article in English | MEDLINE | ID: mdl-10581234

ABSTRACT

The bacterial mismatch-specific uracil-DNA glycosylase (MUG) and eukaryotic thymine-DNA glycosylase (TDG) enzymes form a homologous family of DNA glycosylases that initiate base-excision repair of G:U/T mismatches. Despite low sequence homology, the MUG/TDG enzymes are structurally related to the uracil-DNA glycosylase enzymes, but have a very different mechanism for substrate recognition. We have now determined the crystal structure of the Escherichia coli MUG enzyme complexed with an oligonucleotide containing a non-hydrolysable deoxyuridine analogue mismatched with guanine, providing the first structure of an intact substrate-nucleotide productively bound to a hydrolytic DNA glycosylase. The structure of this complex explains the preference for G:U over G:T mispairs, and reveals an essentially non-specific pyrimidine-binding pocket that allows MUG/TDG enzymes to excise the alkylated base, 3, N(4)-ethenocytosine. Together with structures for the free enzyme and for an abasic-DNA product complex, the MUG-substrate analogue complex reveals the conformational changes accompanying the catalytic cycle of substrate binding, base excision and product release.


Subject(s)
Base Pair Mismatch , DNA Repair , Endodeoxyribonucleases/chemistry , N-Glycosyl Hydrolases/chemistry , Thymine DNA Glycosylase , Catalysis , Crystallography, X-Ray , Cytosine/analogs & derivatives , Cytosine/chemistry , Deoxyribonuclease (Pyrimidine Dimer) , Escherichia coli/enzymology , Models, Molecular , Protein Binding , Protein Conformation , Thymine/chemistry , Uracil/chemistry
17.
Proc Natl Acad Sci U S A ; 96(21): 11809-14, 1999 Oct 12.
Article in English | MEDLINE | ID: mdl-10518532

ABSTRACT

The response of double-helical DNA to torsional stress may be a driving force for many processes acting on DNA. The 1.55-A crystal structure of a duplex DNA oligonucleotide d(CCAGGCCTGG)(2) with an engineered crosslink in the minor groove between the central guanine bases depicts how the duplex can accommodate such torsional stress. We have captured in the same crystal two rather different conformational states. One duplex contains a strained crosslink that is stabilized by calcium ion binding in the major groove, directly opposite the crosslink. For the other duplex, the strain in the crosslink is relieved through partial rupture of a base pair and partial extrusion of a cytosine accompanied by helix bending. The sequence used is the target sequence for the HaeIII methylase, and this partially flipped cytosine is the same nucleotide targeted for extrusion by the enzyme. Molecular dynamics simulations of these structures show an increased mobility for the partially flipped-out cytosine.


Subject(s)
DNA/chemistry , DNA/genetics , Nucleic Acid Conformation , Oligodeoxyribonucleotides/chemistry , Base Pairing , Base Sequence , Calcium/metabolism , Crystallography, X-Ray , Hydrogen Bonding , Models, Molecular , Molecular Sequence Data
18.
J Biol Chem ; 274(29): 20235-43, 1999 Jul 16.
Article in English | MEDLINE | ID: mdl-10400641

ABSTRACT

The high mobility group HMG I(Y) protein has been reported to promote the expression of several NF-kappaB-dependent genes by enhancing the binding of NF-kappaB to DNA. The molecular origins of cooperativity in the binding of NF-kappaB and HMG I(Y) to DNA are not well understood. Here we have examined the determinants of specificity in the binding of HMG I(Y), both alone and in cooperation with NF-kappaB, to two different DNA elements, PRDII from the interferon-beta enhancer and IgkappaB from the immunoglobulin kappa light chain enhancer. Of particular interest was the influence of a flanking AT-rich sequence on binding by HMG I(Y). Utilizing yeast one-hybrid screening assays together with alanine-scanning mutagenesis, we have identified mutations of residues in HMG I(Y) that decrease cooperative binding of NF-kappaB to PRDII and IgkappaB sites. These same mutations similarly decreased the binding of HMG I(Y) alone to DNA, and paradoxically, decreased the strength of protein-protein interactions between HMG I(Y) and NF-kappaB. Of the three tandemly repeated basic regions that represent putative DNA-binding motifs in HMG I(Y), the residues within the second repeat are most important for recognition of core NF-kappaB sites, whereas the second and third repeats both appear to be involved in binding to sites that are flanked by AT-rich sequences. Overall, the second repeat of HMG I(Y) is primarily responsible for the stimulatory effect of this protein on the binding of NF-kappaB to PRDII and IgkappaB elements.


Subject(s)
DNA/metabolism , High Mobility Group Proteins/metabolism , NF-kappa B/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Base Sequence , DNA Primers , HMGA1a Protein , High Mobility Group Proteins/chemistry , High Mobility Group Proteins/genetics , Molecular Sequence Data , Mutagenesis , Protein Binding , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/genetics , Transcription Factors/chemistry , Transcription Factors/genetics
20.
Curr Biol ; 9(4): 174-85, 1999 Feb 25.
Article in English | MEDLINE | ID: mdl-10074426

ABSTRACT

BACKGROUND: The cellular environment exposes DNA to a wide variety of endogenous and exogenous reactive species that can damage DNA, thereby leading to genetic mutations. DNA glycosylases protect the integrity of the genome by catalyzing the first step in the base excision-repair of lesions in DNA. RESULTS: Here, we report a strategy to conduct genome-wide screening for expressed DNA glycosylases, based on their ability to bind to a library of four synthetic inhibitors that target the enzyme's active site. These inhibitors, used in conjunction with the in vitro expression cloning procedure, led to the identification of novel Xenopus and human proteins, xSMUG1 and hSMUG1, respectively, that efficiently excise uracil residues from DNA. Despite a lack of statistically significant overall sequence similarity to the two established classes of uracil-DNA glycosylases, the SMUG1 enzymes contain motifs that are hallmarks of a shared active-site structure and overall protein architecture. The unusual preference of SMUG1 for single-stranded rather than double-stranded DNA suggests a unique biological function in ridding the genome of uracil residues, which are potent endogenous mutagens. CONCLUSIONS: The 'proteomics' approach described here has led to the isolation of a new family of uracil-DNA glycosylases. The three classes of uracil-excising enzymes (SMUG1 being the most recently discovered) represent a striking example of structural and functional conservation in the almost complete absence of sequence conservation.


Subject(s)
DNA Glycosylases , N-Glycosyl Hydrolases/genetics , N-Glycosyl Hydrolases/metabolism , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Cloning, Molecular , Conserved Sequence , Humans , Molecular Sequence Data , N-Glycosyl Hydrolases/chemistry , Protein Conformation , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Uracil-DNA Glycosidase , Xenopus , Xenopus Proteins
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