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1.
BMC Plant Biol ; 23(1): 467, 2023 Oct 06.
Article in English | MEDLINE | ID: mdl-37803262

ABSTRACT

BACKGROUND: The mechanisms and regulation for DNA replication in plant organelles are largely unknown, as few proteins involved in replisome assembly have been biochemically studied. A primase-helicase dubbed Twinkle (T7 gp4-like protein with intramitochondrial nucleoid localization) unwinds double-stranded DNA in metazoan mitochondria and plant organelles. Twinkle in plants is a bifunctional enzyme with an active primase module. This contrast with animal Twinkle in which the primase module is inactive. The organellar primase-helicase of Arabidopsis thaliana (AtTwinkle) harbors a primase module (AtPrimase) that consists of an RNA polymerase domain (RPD) and a Zn + + finger domain (ZFD). RESULTS: Herein, we investigate the mechanisms by which AtTwinkle recognizes its templating sequence and how primer synthesis and coupling to the organellar DNA polymerases occurs. Biochemical data show that the ZFD of the AtPrimase module is responsible for template recognition, and this recognition is achieved by residues N163, R166, and K168. The role of the ZFD in template recognition was also corroborated by swapping the RPDs of bacteriophage T7 primase and AtPrimase with their respective ZFDs. A chimeric primase harboring the ZFD of T7 primase and the RPD of AtPrimase synthesizes ribonucleotides from the T7 primase recognition sequence and conversely, a chimeric primase harboring the ZFD of AtPrimase and the RPD of T7 primase synthesizes ribonucleotides from the AtPrimase recognition sequence. A chimera harboring the RPDs of bacteriophage T7 and the ZBD of AtTwinkle efficiently synthesizes primers for the plant organellar DNA polymerase. CONCLUSIONS: We conclude that the ZFD is responsible for recognizing a single-stranded sequence and for primer hand-off into the organellar DNA polymerases active site. The primase activity of plant Twinkle is consistent with phylogeny-based reconstructions that concluded that Twinkle´s last eukaryotic common ancestor (LECA) was an enzyme with primase and helicase activities. In plants, the primase domain is active, whereas the primase activity was lost in metazoans. Our data supports the notion that AtTwinkle synthesizes primers at the lagging-strand of the organellar replication fork.


Subject(s)
Arabidopsis , DNA Primase , Animals , DNA Primase/genetics , DNA Primase/chemistry , DNA Primase/metabolism , DNA Helicases/chemistry , DNA Helicases/genetics , DNA Helicases/metabolism , DNA-Directed DNA Polymerase/genetics , DNA-Directed DNA Polymerase/metabolism , Arabidopsis/metabolism , Mitochondria/metabolism , Zinc Fingers , Ribonucleotides , DNA Replication , Bacteriophage T7/genetics
2.
J Biomol Struct Dyn ; 41(7): 3062-3075, 2023 04.
Article in English | MEDLINE | ID: mdl-35249470

ABSTRACT

The Rad5 protein is an SWI/SNF family ubiquitin ligase that contains an N-terminal HIRAN domain and a RING C3HC4 motif. The HIRAN domain is critical for recognition of the stalled replication fork during the replication process and acts as a sensor to initiate the damaged DNA checkpoint. It is a conserved domain widely distributed in eukaryotic organisms and is present in several DNA-binding proteins from all kingdoms. Here we showed that distant species have important differences in key residues that affect affinity for ssDNA. Based on these findings, we hypothesized that different HIRAN domains might affect fork reversal and translesion synthesis through different metabolic processes. To address this question, we predicted the tertiary structure of both yeast and human HIRAN domains using molecular modeling. Structural dynamics experiments showed that the yeast HIRAN domain exhibited higher structural denaturation than its human homolog, although both domains became stable in the presence of ssDNA. Analysis of atomic contacts revealed that a greater number of interactions between the ssDNA nucleotides and the Rad5 domain are electrostatic. Taken together, these results provide new insights into the molecular mechanism of the HIRAN domain of Rad5 and may guide us to further elucidate differences in the ancient eukaryotes HIRAN sequences and their DNA affinity.Communicated by Ramaswamy H. Sarma.


Subject(s)
Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Humans , Saccharomyces cerevisiae/genetics , DNA-Binding Proteins/chemistry , DNA Replication , DNA/chemistry , DNA, Single-Stranded , DNA Helicases/chemistry , DNA Helicases/genetics , DNA Helicases/metabolism , Saccharomyces cerevisiae Proteins/genetics
3.
Methods Mol Biol ; 2281: 313-322, 2021.
Article in English | MEDLINE | ID: mdl-33847968

ABSTRACT

Defects in mitochondrial DNA (mtDNA) maintenance may lead to disturbances in mitochondrial homeostasis and energy production in eukaryotic cells, causing diseases. During mtDNA replication, the mitochondrial single-stranded DNA-binding protein (mtSSB) stabilizes and protects the exposed single-stranded mtDNA from nucleolysis; perhaps more importantly, it appears to coordinate the actions of both the replicative mtDNA helicase Twinkle and DNA polymerase gamma at the replication fork. Here, we describe a helicase stimulation protocol to test in vitro the functional interaction between mtSSB and variant forms of Twinkle. We show for the first time that the C-terminal tail of Twinkle is important for such an interaction, and that it negatively regulates helicase unwinding activity in a salt-dependent manner.


Subject(s)
DNA Helicases/chemistry , DNA Helicases/metabolism , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/metabolism , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/metabolism , Mutation , Binding Sites , DNA Helicases/genetics , DNA Replication , DNA, Mitochondrial/chemistry , DNA, Mitochondrial/metabolism , DNA, Single-Stranded/chemistry , DNA-Binding Proteins/chemistry , Humans , Mitochondrial Proteins/genetics , Models, Molecular , Protein Binding , Protein Conformation
4.
Arch Biochem Biophys ; 703: 108841, 2021 05 30.
Article in English | MEDLINE | ID: mdl-33775623

ABSTRACT

ATPases belonging to the AAA+ superfamily are associated with diverse cellular activities and are mainly characterized by a nucleotide-binding domain (NBD) containing the Walker A and Walker B motifs. AAA+ proteins have a range of functions, from DNA replication to protein degradation. Rvbs, also known as RUVBLs, are AAA+ ATPases with one NBD domain and were described from human to yeast as participants of the R2TP (Rvb1-Rvb2-Tah1-Pih1) complex. Although essential for the assembly of multiprotein complexes-containing DNA and RNA, the protozoa Rvb orthologs are less studied. For the first time, this work describes the Rvbs from Leishmania major, one of the causative agents of Tegumentar leishmaniasis in human. Recombinant LmRUVBL1 and LmRUVBL2 his-tagged proteins were successfully purified and investigated using biophysical tools. LmRUVBL1 was able to form a well-folded elongated hexamer in solution, while LmRUVBL2 formed a large aggregate. However, the co-expression of LmRUVBL1 and LmRUVBL2 assembled the proteins into an elongated heterodimer in solution. Thermo-stability and fluorescence experiments indicated that the LmRUVBL1/2 heterodimer had ATPase activity in vitro. This is an interesting result because hexameric LmRUVBL1 alone had low ATPase activity. Additionally, using independent SL-RNAseq libraries, it was possible to show that both proteins are expressed in all L. major life stages. Specific antibodies obtained against LmRUVBLs identified the proteins in promastigotes and metacyclics cell extracts. Together, the results here presented are the first step towards the characterization of Leishmania Rvbs, and may contribute to the development of possible strategies to intervene against leishmaniasis, a neglected tropical disease of great medical importance.


Subject(s)
Adenosine Triphosphatases/metabolism , DNA Helicases/chemistry , DNA Helicases/metabolism , Leishmania major/enzymology , Protein Multimerization , Amino Acid Sequence , Protein Folding , Protein Structure, Quaternary , Solutions
5.
Parasite ; 21: 7, 2014.
Article in English | MEDLINE | ID: mdl-24534563

ABSTRACT

Entamoeba histolytica, the protozoan responsible for human amoebiasis, exhibits a great genome plasticity that is probably related to homologous recombination events. It contains the RAD52 epistasis group genes, including Ehrad51 and Ehrad54, and the Ehblm gene, which are key homologous recombination factors in other organisms. Ehrad51 and Ehrad54 genes are differentially transcribed in trophozoites when DNA double-strand breaks are induced by ultraviolet-C irradiation. Moreover, the EhRAD51 recombinase is overexpressed at 30 min in the nucleus. Here, we extend our analysis of the homologous recombination mechanism in E. histolytica by studying EhRAD51, EhRAD54, and EhBLM expression in response to DNA damage. Bioinformatic analyses show that EhRAD54 has the molecular features of homologous proteins, indicating that it may have similar functions. Western blot assays evidence the differential expression of EhRAD51, EhRAD54, and EhBLM at different times after DNA damage, suggesting their potential roles in the different steps of homologous recombination in this protozoan.


Subject(s)
DNA Repair , Entamoeba histolytica/metabolism , Homologous Recombination , Protozoan Proteins/physiology , Amino Acid Sequence , Animals , Cell Nucleus/chemistry , Consensus Sequence , Cytoplasm/chemistry , DNA Breaks, Double-Stranded , DNA Helicases/chemistry , DNA Helicases/genetics , DNA Helicases/physiology , DNA Repair/genetics , DNA, Protozoan/genetics , DNA, Protozoan/radiation effects , Entamoeba histolytica/genetics , Entamoeba histolytica/radiation effects , Genes, Protozoan , Homologous Recombination/genetics , Humans , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Rad51 Recombinase/genetics , Rad51 Recombinase/physiology , RecQ Helicases/genetics , RecQ Helicases/physiology , Sequence Alignment , Sequence Homology, Amino Acid , Time Factors , Ultraviolet Rays
6.
Mol Genet Genomics ; 274(1): 79-90, 2005 Aug.
Article in English | MEDLINE | ID: mdl-15931527

ABSTRACT

In most filamentous fungi, telomere-associated sequences (TASs) are polymorphic, and the presence of restriction fragment length polymorphisms (RFLPs) may permit the number of chromosome ends to be estimated from the number of telomeric bands obtained by restriction digestion. Here, we describe strains of Metarhizium, Gliocladium and Paecilomyces species in which only one or a few telomeric bands of unequal intensity are detectable by Southern hybridization, indicating that interchromosomal TAS exchange occurs. We also studied an anomalous strain of Metarhizium anisopliae, which produces polymorphic telomeric bands larger than 8 kb upon digestion of genomic DNA with XhoI. In this case, the first XhoI site in from the chromosome end must lie beyond the presumed monomorphic region. Cloned telomeres from this strain comprise 18-26 TTAGGG repeats, followed at the internal end of the telomere tract by five repeats of the telomere-like sequence TAAACGCTGG. An 8.1-kb TAS clone also contains a gene for a RecQ-like helicase, designated TAH1, suggesting that this TAS is analogous to the Y' elements in yeast and the subtelomeric helicase ORFs of Ustilago maydis (UTASRecQ) and Magnaporthe grisea (TLH1). The TAS in the anomalous strain of M. anisopliae, however, appears distinct from these in that it is found at most telomeres and its predicted protein product possesses a significantly longer N-terminal region in comparison to the M. grisea and U. maydis helicases. Hybridization analyses showed that TAH1 homologues are present in all other anomalous M. anisopliae strains studied, as well as in some other polymorphic strains, where the recQ-like gene also appears to be telomere-associated.


Subject(s)
Fungal Proteins/genetics , Fungal Proteins/metabolism , Hypocreales/metabolism , Telomere/physiology , Adenosine Triphosphatases/chemistry , Amino Acid Sequence , Blotting, Northern , Blotting, Southern , Chromosomes, Fungal/genetics , Chromosomes, Fungal/metabolism , Cloning, Molecular , DNA Helicases/chemistry , DNA, Fungal/genetics , DNA, Fungal/metabolism , Hypocreales/classification , Hypocreales/genetics , Molecular Sequence Data , Phylogeny , Polymorphism, Genetic , Polymorphism, Restriction Fragment Length , RecQ Helicases , Sequence Homology, Amino Acid
7.
Biochem Biophys Res Commun ; 313(4): 834-40, 2004 Jan 23.
Article in English | MEDLINE | ID: mdl-14706617

ABSTRACT

Plasmid R6K-encoded pi protein has multiple regulatory functions in replication and transcription. These functions rely, in part, on a complex set of interactions between monomers and dimers of the protein and distinct DNA targets, the direct and inverted repeats (DRs, IRs). In the work described here, we examine the isomerization and DNA bending properties of pi using electrophoretic mobility shift assays and circular permutation assays. Our data suggest that pi dimers can bend IRs, and dimer subunits seem to readily associate in head-to-head and head-to-tail fashion. The ability of pi to bend DRs is also reexamined using techniques that allow us to discriminate between bending induced by its different isomeric forms. We find that both monomers and dimers bend a single DR to similar degrees while results with 2DRs are more complex. The significance of the bending data in regard to a possible mechanism for replication initiation by pi protein is discussed.


Subject(s)
DNA Helicases/metabolism , DNA, Bacterial/chemistry , DNA-Binding Proteins , Escherichia coli Proteins/metabolism , Plasmids/chemistry , Trans-Activators/metabolism , DNA Helicases/chemistry , DNA Helicases/genetics , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Models, Molecular , Nucleic Acid Conformation , Plasmids/genetics , Plasmids/metabolism , Repetitive Sequences, Nucleic Acid , Trans-Activators/chemistry , Trans-Activators/genetics
8.
Clin Biochem ; 36(1): 41-9, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12554059

ABSTRACT

It has been shown that the Hepatitis C virus nonstructural NS3 protein possesses at least two enzymatic domains: a serine-protease domain and an adenosine triphosphatase (ATPase)/helicase domain. In this report, a truncated fragment of NS3 (26 kDa), representing main epitopes from the (ATPase)/helicase domain, has been expressed in Escherichia coli. The recombinant protein was purified by Ion Metal Affinity Chromatography (IMAC) with more than 90% purity. The recognition of B-cell linear epitopes in the NS3 protein was evaluated by immunoblot. The recombinant NS3 protein was reduced and carboxymethylated, and the recognition of either conformational and/or linear B-cell determinants was evaluated by ELISA. The inclusion of the recombinant NS3 protein in a third-generation diagnostic system UltraMicroELISA (UMELISA) allowed an increase in the sensitivity, due to the detection of a new variety of false-negative sera in blood donor test samples.


Subject(s)
Adenosine Triphosphatases/chemistry , DNA Helicases/chemistry , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Chromatography, Affinity , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Epitopes , Escherichia coli/metabolism , Fermentation , Immunoblotting , Models, Genetic , Plasmids/metabolism , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Temperature
9.
FEBS Lett ; 533(1-3): 14-20, 2003 Jan 02.
Article in English | MEDLINE | ID: mdl-12505151

ABSTRACT

The two human proteins Ki-1/57 and CGI-55 have highly similar amino acid sequences but their functions are unknown. We analyzed them by yeast two-hybrid screens and found that they interact with the C-terminal region of the human chromatin-remodeling factor CHD-3 (chromo-helicase-DNA-binding domain protein-3). The interaction of CGI-55 and CHD-3 could be confirmed in vitro and in vivo by co-immunoprecipitations from Sf9 insect cells. Mapping showed that CGI-55 interacts with CHD-3 via two regions at its N- and C-terminals. The CGI-55 and Ki-1/57 mRNAs show highest expression in muscle, colon and kidney. A CGI55-GFP fusion protein was localized in the cytoplasm, nucleus and perinuclear regions of HeLa cells. These data suggest the possibility that CGI-55 and Ki-1/57 might be involved in nuclear functions like the remodeling of chromatin.


Subject(s)
Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , DNA Helicases/chemistry , DNA Helicases/metabolism , Hyaluronan Receptors/chemistry , Hyaluronan Receptors/metabolism , Ki-1 Antigen/chemistry , Ki-1 Antigen/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Adenosine Triphosphatases/genetics , Amino Acid Sequence , Animals , Binding Sites , Caenorhabditis elegans Proteins/chemistry , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Chromatin/metabolism , DNA Helicases/genetics , Gene Expression , HeLa Cells , Humans , Hyaluronan Receptors/genetics , In Vitro Techniques , Ki-1 Antigen/genetics , Mi-2 Nucleosome Remodeling and Deacetylase Complex , Molecular Sequence Data , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Tissue Distribution
10.
Gene ; 208(2): 207-13, 1998 Feb 27.
Article in English | MEDLINE | ID: mdl-9524267

ABSTRACT

The human gene XPB, defective in xeroderma pigmentosum patients complementation group B, encodes a DNA helicase involved in several DNA metabolic pathways, including DNA repair and transcription. The high conservation of this gene has allowed the cloning of homologs in various species, such as mouse, yeast and Drosophila. Not much information on the molecular basis of nucleotide excision repair in plants is available, but these organisms may have similar mechanisms to other eukaryotes. A homolog of XPB was isolated in Arabidopsis thaliana by using polymerase chain reaction (PCR) with degenerate oligonucleotides based on protein domains which are conserved among several species. Screening of an Arabidopsis cDNA library led to the identification and isolation of a cDNA clone with 2670 bp encoding a predicted protein of 767 amino acids, denoted araXPB. Genomic analysis indicated that this is a nuclear single copy gene in plant cells. Northern blot with the cDNA probe revealed a major transcript which migrated at approx. 2,800 b, in agreement with the size of the cDNA isolated. The araXPB protein shares approximately 50% identical and 70% conserved amino acids with the yeast and human homologs. The plant protein maintains all the functional domains found in the other proteins, including nuclear localization signal, DNA-binding domain and helicase motifs, suggesting that it might also act as part of the RNA transcription apparatus, as well as nucleotide excision repair in plant cells.


Subject(s)
Arabidopsis/genetics , Arabidopsis/metabolism , DNA Helicases/genetics , DNA-Binding Proteins/genetics , Amino Acid Sequence , Animals , Cloning, Molecular , Conserved Sequence , DNA Helicases/chemistry , DNA, Complementary , DNA, Plant/genetics , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/chemistry , Drosophila/genetics , Genes, Plant , Humans , Mice , Molecular Sequence Data , Polymerase Chain Reaction , Saccharomyces cerevisiae/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Xeroderma Pigmentosum/genetics
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