An evolutionary analysis of the helix-hairpin-helix superfamily of DNA repair glycosylases

The helix-hairpin-helix (HhH) superfamily of base excision repair DNA glycosylases is composed of multiple phylogenetically diverse enzymes that are capable of excising varying spectra of oxidatively and methyl-damaged bases. Although these DNA repair glycosylases have been widely studied through genetic, biochemical, and biophysical approaches, the evolutionary relationships of different HhH homologs and the extent to which they are conserved across phylogeny remain enigmatic. We provide an evolutionary framework for this pervasive and versatile superfamily of DNA glycosylases. Six HhH gene families (named AlkA: alkyladenine glycosylase; MpgII: N-methylpurine glycosylase II; MutY/Mig: A/G-specific adenine glycosylase/mismatch glycosylase; Nth: endonuclease III; OggI: 8-oxoguanine glycosylase I; and OggII: 8-oxoguanine glycosylase II) are identified through phylogenetic analysis of 234 homologs found in 94 genomes (16 archaea, 64 bacteria, and 14 eukaryotes). The number of homologs in each gene family varies from 117 in the Nth family (nearly every genome surveyed harbors at least one Nth homolog) to only five in the divergent OggII family (all from archaeal genomes). Sequences from all three domains of life are included in four of the six gene families, suggesting that the HhH superfamily diversified very early in evolution. The phylogeny provides evidence for multiple lineage-specific gene duplication events, most of which involve eukaryotic homologs in the Nth and AlkA gene families. We observe extensive variation in the number of HhH superfamily glycosylase genes present in different genomes, possibly reflecting major differences among species in the mechanisms and pathways by which damaged bases are repaired and/or disparities in the basic rates and spectra of mutation experienced by different genomes.     

A Leuconostoc lactis protein with homology to ribosomal protein S1 shares common epitopes and common DNA binding properties with a mammalian DNA binding nuclear factor

A mouse testis cDNA expression library (Clontech) was screened with a synthetic oligonucleotide ligand containing CT-rich motifs derived from the rat skeletal muscle actin gene promoter. These motifs bind nuclear proteins, and seem to be involved in the regulation of the gene. Analysis of isolated clones, which expressed proteins that specifically bind the oligonucleotide, indicated that they were derived from a single gene. This gene was identified as a contaminant of bacterial origin (Leuconostoc lactis). The cloned gene from L. lactis encodes a protein with significant homology to bacterial ribosomal protein S1, which we designated LrpS1-L. Band shift analysis and competition experiments indicated that both the bacterial protein and a mouse nuclear protein specifically bind to the same CT-rich motif of the skeletal muscle actin promoter. Furthermore, antibodies against the recombinant bacterial protein interfered with the formation of complex between the CT-rich element and the mouse nuclear protein. These results indicate that the bacterial LrpS1-L protein and the mammalian protein bind the same CT-rich motif and share common antigenic epitopes.     

Hormonal regulation of a chicken oviduct messenger ribonucleic acid that shares a common domain with gizzard myosin light chain kinase

Changes in myosin light chain kinase (MLCK) and calmodulin (CaM) mRNAs have been evaluated during estrogen-mediated differentiation of the chicken oviduct. Also examined were acute changes that occur in oviduct RNA from animals stimulated with estrogen, withdrawn from hormone and then injected for 1, 2, and 4 days with synthetic estrogen [diethylstilbestrol (DES)], progesterone (P), or testosterone (T). Small changes were noted in both CaM and MLCK RNAs during primary stimulation when oviduct cells are actively dividing. On the other hand no significant changes were observed during secondary stimulation regardless of the steroid hormone injected. These data support the contention that CaM and MLCK are constitutively expressed but vary as a function of cell cycle. The MLCK mRNA is 5.5 kilobases (kb) but the MLCK cDNA also hybridizes to an oviduct RNA 2.7 kb long. This RNA species is acutely regulated by estrogen, P, and T but in a manner different from that of ovalbumin mRNA. The magnitude of stimulation of the 2.7 kb mRNA by diethylstilbestrol and T is greater than that of ovalbumin whereas changes in response to P are similar. The 12- to 16-fold increase of the 2.7 kb mRNA in response to T is the largest effect reported for this hormone acting on oviduct. The 2.7 kb mRNA encodes an unknown protein yet contains a 520 nucleotide segment that is highly homologous with the COOH-terminal coding portion of the MLCK mRNA. Since this homology does not include either catalytic or CaM-binding domains of MLCK, it is unlikely that the 2.7 kb mRNA encodes a CaM-dependent protein kinase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural characterization of the Fpg family of DNA glycosylases

Until recently, the Fpg family was the only major group of DNA glycosylases for which no structural data existed. Prototypical members of this family, found in eukaryotes as well as prokaryotes, have now been crystallized as free proteins and as complexes with DNA. In this review, we analyze the available structural information for formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease VIII (Nei). Special emphasis is placed on mechanisms by which these enzymes recognize and selectively excise cognate lesions from oxidatively damaged DNA. The problem of lesion recognition is considered in two parts: how the enzyme efficiently locates a single lesion embedded in a vast excess of DNA; and how the lesion is accommodated in a pocket near the active site of the enzyme. Although all crystal structures reported to date for the Fpg family lack the damaged base, functionally important residues that participate in DNA binding and enzyme catalysis have been clearly identified and other residues, responsible for substrate specificity, have been inferred.     

A novel uracil-DNA glycosylase family related to the helix-hairpin-helix DNA glycosylase superfamily

Cytosine bases can be deaminated spontaneously to uracil, causing DNA damage. Uracil-DNA glycosylase (UDG), a ubiquitous uracil-excising enzyme found in bacteria and eukaryotes, is one of the enzymes that repair this kind of DNA damage. To date, no UDG-coding gene has been identified in Methanococcus jannaschii, although its entire genome was deciphered. Here, we have identified and characterized a novel UDG from M.jannaschii designated as MjUDG. It efficiently removed uracil from both single- and double-stranded DNA. MjUDG also catalyzes the excision of 8-oxoguanine from DNA. MjUDG has a helix–hairpin–helix motif and a [4Fe–4S]-binding cluster that is considered to be important for the DNA binding and catalytic activity. Although MjUDG shares these features with other structural families such as endonuclease III and mismatch-specific DNA glycosylase (MIG), unique conserved amino acids and substrate specificity distinguish MjUDG from other families. Also, a homologous member of MjUDG was identified in Aquifex aeolicus. We report that MjUDG belongs to a novel UDG family that has not been described to date.     

The alpha/beta fold uracil DNA glycosylases: a common origin with diverse fates

Background  

Uracil DNA glycosylases (UDGs) are major repair enzymes that protect DNA from mutational damage caused by uracil incorporated as a result of a polymerase error or deamination of cytosine. Four distinct families of UDGs have been identified, which show very limited sequence similarity to each other, although two of them have been shown to possess the same structural fold. The structural and evolutionary relationships between the rest of the UDGs remain uncertain.     

MAN1, an inner nuclear membrane protein that shares the LEM domain with lamina-associated polypeptide 2 and emerin

The "MAN antigens" are polypeptides recognized by autoantibodies from a patient with a collagen vascular disease and localized to the nuclear envelope. We now show that one of the human MAN antigens termed MAN1 is a 82.3-kDa protein with an amino-terminal domain followed by two hydrophobic segments and a carboxyl-terminal tail. The MAN1 gene contains seven protein-coding exons and is assigned to human chromosome 12q14. Its mRNA is approximately 5.5 kilobases and is detected in several different cell types that were examined. Cell extraction experiments show that MAN1 is an integral membrane protein. When expressed in transfected cells, MAN1 is exclusively targeted to the nuclear envelope, consistent with an inner nuclear membrane localization. Protein sequence analysis reveals that MAN1 shares a conserved globular domain of approximately 40 amino acids, which we term the LEM module, with inner nuclear membrane proteins lamina-associated polypeptide 2 and emerin. The LEM module is also present in two proteins of Caenorhabditis elegans. These results show that MAN1 is an integral protein of the inner nuclear membrane that shares the LEM module with other proteins of this subcellular localization.     

The dermaseptin precursors: a protein family with a common preproregion and a variable C-terminal antimicrobial domain.

Preprodermaseptins are a group of antimicrobial peptide precursors found in the skin of a variety of frog species. Precursors of this family have very similar N-terminal preprosequences followed by markedly different C-terminal domains that correspond to mature antimicrobial peptides. Some of these peptides are 24-34 amino acids long and form well-behaved amphipathic alpha-helices, others are disulfide-linked peptides of 20-46 residues, still others, highly hydrophobic, are the smallest antimicrobial peptides known so far being only 10-13 residues in length. All these peptides are broad-spectrum microbicides that kill many bacteria, protozoa, yeasts and fungi by destroying or permeating the microbial membrane. In frogs belonging to the genus Phyllomedusinae, preprodermaseptins encoded peptides also include dermorphins and deltorphins, D-amino acid-containing heptapeptides which are very potent and specific agonists of the mu- or delta-opioid receptors. The remarkable similarity between preproregions of precursors that give rise to peptides with very different primary structures, conformations and activities suggests that the corresponding genes originate from a common ancestor. The high conservation of the precursor prepropart indicates that this region must have an important function.     

A discontinuous DNA glycosylase domain in a family of enzymes that excise 5-methylcytosine

DNA cytosine methylation (5-meC) is a widespread epigenetic mark associated to gene silencing. In plants, DEMETER-LIKE (DML) proteins typified by Arabidopsis REPRESSOR OF SILENCING 1 (ROS1) initiate active DNA demethylation by catalyzing 5-meC excision. DML proteins belong to the HhH-GPD superfamily, the largest and most functionally diverse group of DNA glycosylases, but the molecular properties that underlie their capacity to specifically recognize and excise 5-meC are largely unknown. We have found that sequence similarity to HhH-GPD enzymes in DML proteins is actually distributed over two non-contiguous segments connected by a predicted disordered region. We used homology-based modeling to locate candidate residues important for ROS1 function in both segments, and tested our predictions by site-specific mutagenesis. We found that amino acids T606 and D611 are essential for ROS1 DNA glycosylase activity, whereas mutations in either of two aromatic residues (F589 and Y1028) reverse the characteristic ROS1 preference for 5-meC over T. We also found evidence suggesting that ROS1 uses Q607 to flip out 5-meC, while the contiguous N608 residue contributes to sequence-context specificity. In addition to providing novel insights into the molecular basis of 5-meC excision, our results reveal that ROS1 and its DML homologs possess a discontinuous catalytic domain that is unprecedented among known DNA glycosylases.     

Heavy metal ions affect the activity of DNA glycosylases of the Fpg family

Prokaryotic enzymes formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease VIII (Nei) and their eukaryotic homologs NEIL1, NEIL2, and NEIL3 define the Fpg family of DNA glycosylases, which initiate the process of repair of oxidized DNA bases. The repair of oxidative DNA lesions is known to be impaired in vivo in the presence of ions of some heavy metals. We have studied the effect of salts of several alkaline earth and transition metals on the activity of Fpg-family DNA glycosylases in the reaction of excision of 5,6-dihydrouracil, a typical DNA oxidation product. The reaction catalyzed by NEIL1 was characterized by values K _m = 150 nM and k _cat = 1.2 min⁻¹, which were in the range of these constants for excision of other damaged bases by this enzyme. NEIL1 was inhibited by Al³⁺, Ni²⁺, Co²⁺, Cd²⁺, Cu²⁺, Zn²⁺, and Fe²⁺ in Tris-HCl buffer and by Cd²⁺, Zn²⁺, Cu²⁺, and Fe²⁺ in potassium phosphate buffer. Fpg and Nei, the prokaryotic homologs of NEIL1, were inhibited by the same metal ions as NEIL1. The values of I₅₀ for NEIL1 inhibition were 7 μM for Cd²⁺, 16 μM for Zn²⁺, and 400 μM for Cu²⁺. The inhibition of NEIL1 by Cd²⁺, Zn²⁺, and Cu²⁺ was at least partly due to the formation of metal-DNA complexes. In the case of Cd²⁺ and Cu²⁺, which preferentially bind to DNA bases rather than phosphates, the presence of metal ions caused the enzyme to lose the ability for preferential binding to damaged DNA. Therefore, the inhibition of NEIL1 activity in removal of oxidative lesions by heavy metal ions may be a reason for their comutagenicity under oxidative stress.     

SAF-Box, a conserved protein domain that specifically recognizes scaffold attachment region DNA

SARs (scaffold attachment regions) are candidate DNA elements for partitioning eukaryotic genomes into independent chromatin loops by attaching DNA to proteins of a nuclear scaffold or matrix. The interaction of SARs with the nuclear scaffold is evolutionarily conserved and appears to be due to specific DNA binding proteins that recognize SARs by a mechanism not yet understood. We describe a novel, evolutionarily conserved protein domain that specifically binds to SARs but is not related to SAR binding motifs of other proteins. This domain was first identified in human scaffold attachment factor A (SAF-A) and was thus designated SAF-Box. The SAF-Box is present in many different proteins ranging from yeast to human in origin and appears to be structurally related to a homeodomain. We show here that SAF-Boxes from four different origins, as well as a synthetic SAF-Box peptide, bind to natural and artificial SARs with high specificity. Specific SAR binding of the novel domain is achieved by an unusual mass binding mode, is sensitive to distamycin but not to chromomycin, and displays a clear preference for long DNA fragments. This is the first characterization of a specific SAR binding domain that is conserved throughout evolution and has DNA binding properties that closely resemble that of the unfractionated nuclear scaffold.     

A 165 kd protein of the herpes simplex virion shares a common epitope with the regulatory protein, ICP4

We have investigated the possibility that immediate-early (IE) protein ICP4 could be a part of herpes simplex virus type 1 (HSV-1) virion particle. Immunodetection with a monoclonal antibody against ICP4 reveals that a component of the virion, migrating at 165 kd, shares a common epitope with this immediate-early protein. Immunolocalization studies on purified virions indicate that the antigen can be detected only in virions without membranes, and is located outside the capsid, most probably in the tegument. Ultrastructural localizations on HSV-1 infected BHK cells extracted with a nonionic detergent confirm that the protein immunoreacting with anti-ICP4 is present in virions.     

Eukaryotic RNAse H shares a conserved domain with caulimovirus proteins that facilitate translation of polycistronic RNA.

RNAse H (RNH1 protein) from the trypanosomatid Crithidia fasciculata has a functionally uncharacterized N-terminal domain dispensable for the RNAse H activity. Using computer methods for database search and multiple alignment, we show that the N-terminal domains of RNH1 and its homologue encoded by a cDNA from chicken lens are related to the conserved domain in caulimovirus ORF VI product that facilitates translation of polycistronic virus RNA in plant cells. We hypothesize that the N-terminal domain of eukaryotic RNAse H performs an as yet uncharacterized regulatory function, possibly in mRNA translation or turnover.     

Cloning and subcellular location of an Arabidopsis receptor-like protein that shares common features with protein-sorting receptors of eukaryotic cells.

Many receptors involved in clathrin-mediated protein transport through the endocytic and secretory pathways of yeast and animal cells share common features. They are all type I integral membrane proteins containing cysteine-rich lumenal domains and cytoplasmic tails with tyrosine-containing sorting signals. The cysteine-rich domains are thought to be involved in ligand binding, whereas the cytoplasmic tyrosine motifs interact with clathrin-associated adaptor proteins during protein sorting along these pathways. In addition, tyrosine-containing signals are required for the retention and recycling of some of these membrane proteins to the trans-Golgi network. Here we report the characterization of an approximately 80-kD epidermal growth factor receptor-like type I integral membrane protein containing all of these functional motifs from Arabidopsis thaliana (called AtELP for A. thaliana Epidermal growth factor receptor-Like Protein). Biochemical analysis indicates that AtELP is a membrane protein found at high levels in the roots of both monocots and dicots. Subcellular fractionation studies indicate that the AtELP protein is present in two membrane fractions corresponding to a novel, undefined compartment and a fraction enriched in vesicles containing clathrin and its associated adaptor proteins. AtELP may therefore serve as a marker for compartments involved in intracellular protein trafficking in the plant cell.     

A cold-regulated nucleic acid-binding protein of winter wheat shares a domain with bacterial cold shock proteins

The molecular mechanisms of cold acclimation are still largely unknown; however, it has been established that overwintering plants such as winter wheat increases freeze tolerance during cold treatments. In prokaryotes, cold shock proteins are induced by temperature downshifts and have been proposed to function as RNA chaperones. A wheat cDNA encoding a putative nucleic acid-binding protein, WCSP1, was isolated and found to be homologous to the predominant CspA of Escherichia coli. The putative WCSP1 protein contains a three-domain structure consisting of an N-terminal cold shock domain with two internal conserved consensus RNA binding domains and an internal glycine-rich region, which is interspersed with three C-terminal CX(2)CX(4)HX(4)C (CCHC) zinc fingers. Each domain has been described independently within several nucleotide-binding proteins. Northern and Western blot analyses showed that WCSP1 mRNA and protein levels steadily increased during cold acclimation, respectively. WCSP1 induction was cold-specific because neither abscisic acid treatment, drought, salinity, nor heat stress induced WCSP1 expression. Nucleotide binding assays determined that WCSP1 binds ssDNA, dsDNA, and RNA homopolymers. The capacity to bind dsDNA was nearly eliminated in a mutant protein lacking C-terminal zinc fingers. Structural and expression similarities to E. coli CspA suggest that WCSP1 may be involved in gene regulation during cold acclimation.     

Characterization of a Drosophila centrosome protein CP309 that shares homology with Kendrin and CG-NAP

The centrosome in animal cells provides a major microtubule-nucleating site that regulates the microtubule cytoskeleton temporally and spatially throughout the cell cycle. We report the identification in Drosophila melanogaster of a large coiled-coil centrosome protein that can bind to calmodulin. Biochemical studies reveal that this novel Drosophila centrosome protein, centrosome protein of 309 kDa (CP309), cofractionates with the gamma-tubulin ring complex and the centrosome-complementing activity. We show that CP309 is required for microtubule nucleation mediated by centrosomes and that it interacts with the gamma-tubulin small complex. These findings suggest that the microtubule-nucleating activity of the centrosome requires the function of CP309.     

Defining a novel domain that provides an essential contribution to site-specific interaction of Rep protein with DNA

An essential feature of replication initiation proteins is their ability to bind to DNA. In this work, we describe a new domain that contributes to a replication initiator sequence-specific interaction with DNA. Applying biochemical assays and structure prediction methods coupled with DNA–protein crosslinking, mass spectrometry, and construction and analysis of mutant proteins, we identified that the replication initiator of the broad host range plasmid RK2, in addition to two winged helix domains, contains a third DNA-binding domain. The phylogenetic analysis revealed that the composition of this unique domain is typical within the described TrfA-like protein family. Both in vitro and in vivo experiments involving the constructed TrfA mutant proteins showed that the newly identified domain is essential for the formation of the protein complex with DNA, contributes to the avidity for interaction with DNA, and the replication activity of the initiator. The analysis of mutant proteins, each containing a single substitution, showed that each of the three domains composing TrfA is essential for the formation of the protein complex with DNA. Furthermore, the new domain, along with the winged helix domains, contributes to the sequence specificity of replication initiator interaction within the plasmid replication origin.     

A family of proteins containing a conserved domain that mediates interaction with the yeast SNF1 protein kinase complex.

The SNF1 protein kinase is required for the regulatory response to glucose starvation in Saccharomyces cerevisiae. SNF1 is a protein serine/threonine kinase that has been widely conserved in both plants and mammals. Previously, we identified SIP1 and SIP2 as proteins that interact with SNF1 in vivo by the two-hybrid system. We have cloned the SIP2 gene and the encoded protein is homologous to SIP1 and to GAL83, which affects glucose repression of the GAL genes. We show that SIP2 and GAL83, like SIP1, co-immunoprecipitate with SNF1 and are phosphorylated in vitro. An 80 amino acid sequence, designated the ASC domain, is highly conserved at the C-termini of all three proteins. We show that this small domain can mediate protein-protein interaction with the SNF1 kinase complex. Thus, SIP1, SIP2 and GAL83 define a family of homologous proteins that are tightly associated with the SNF1 kinase, probably in alternative forms of the complex. Genetic evidence suggests that the three proteins have distinct, but related, functions in the SNF1 pathway, and deletion of GAL83 dramatically reduces SNF1 activity in immune complex assays. We propose that SIP1, SIP2 and GAL83 act as adaptors that promote the activity of SNF1 towards specific targets.