The main regulatory region of mammalian mitochondrial DNA: Structure-function model and evolutionary pattern

Summary The evolution of the main regulatory region (D-loop) of the mammalian mitochondrial genome was analyzed by comparing the sequences of eight mammalian species: human, common chimpanzee, pygmy chimpanzee, dolphin, cow, rat, mouse, and rabbit. The best alignment of the sequences was obtained by optimization of the sequence similarities common to all these species.The two peripheral left and right D-loop domains, which contain the main regulatory elements so far discovered, evolved rapidly in a species-specific manner generating heterogeneity in both length and base composition. They are prone to the insertion and deletion of elements and to the generation of short repeats by replication slippage. However, the preservation of some sequence blocks and similar cloverleaf-like structures in these regions, indicates a basic similarity in the regulatory mechanisms of the mitochondrial genome in all mammalian species.We found, particularly in the right domain, significant similarities to the telomeric sequences of the mitochondrial (mt) and nuclear DNA ofTetrahymena thermophila. These sequences may be interpreted as relics of telomeres present in ancestral linear forms of mtDNA or may simply represent efficient templates of RNA primase-like enzymes.Due to their peculiar evolution, the two peripheral domains cannot be used to estimate in a quantitative way the genetic distances between mammalian species. On the other hand the central domain, highly conserved during evolution, behaves as a good molecular clock.Reliable estimates of the times of divergence between closely and distantly related species were obtained from the central domain using a Markov model and assuming nonhomogeneous evolution of nucleotide sites.     

Sequence-dependent DNA curvature: conformational signal present in the main regulatory region of the rat mitochondrial genome.

Theoretical analysis and experimental approaches by gel electrophoresis in retarding conditions allowed us to identify the presence of an intrinsic bending in the D-loop containing region of the rat mitochondrial genome. The curvature was located in the right domain of the sequence analyzed, between the origin of replication of the heavy strand and its promoter. The preliminary evidence of a specific recognition of the bent DNA with mitochondrial matrix proteins suggests a probable role of this DNA conformation in the duplication and/or expression of the mammalian mitochondrial genome.     

Initiation of DNA replication in eukaryotes is an intriguing cascade of protein interactions

Initiation of eukaryotic DNA replication is a complex process including the recognition of initiation sites on DNA, multi-step DNA preparation for duplication, and assembly of multi-protein complexes capable of beginning DNA synthesis at initiation sites. The process starts at the late M phase and lasts till the appropriate time of the S phase for each initiation site. A chain of interesting interactions between Orc1p-6p, Cdc6p, Mcm2p-7p, Mcm10p, Cdt1, Cdc45p, Dbf4/Cdc7p, RPA, and DNA polymerase takes place during this period. The sequence of these interactions is controlled by cyclin-dependent kinases, as well as by ubiquitin-dependent proteolysis in the proteasome. This review summarizes the data on proteins initiating DNA replication and factors controlling their activities.     

Use of electrospray ionization mass spectrometry to study binding interactions between a replication terminator protein and DNA

Tus protein binds tightly to specific DNA sequences (Ter) on the Escherichia coli chromosome halting replication. We report here conditions for detecting the 1 : 1 Tus-Ter complex by electrospray ionization mass spectrometry (ESI-MS). ESI mass spectra of a mixture of Tus and nonspecific DNA showed ions predominantly from uncomplexed Tus protein, indicating that the Tus-Ter complex observed in the gas phase was the result of a specific interaction rather than nonspecific associations in the ionization source. The Tus-Ter complex was very stable using a spray solvent of 10 mM ammonium acetate at pH 8.0, and initial attempts to distinguish binding affinities of Tus and mutant Tus proteins for Ter DNA were unsuccessful. Increasing the ammonium acetate concentration in the electrospray solvent (800 mM at pH 8.0) increased the dissociation constants sufficiently such that relative orders of binding affinity for Tus and various mutant Tus proteins for various DNA sequences could be determined. These were in agreement with the dissociation constants determined in solution studies. A dissociation constant of 700 x 10(-9) M for the binding of the mutant Tus protein A173T (where residue 173 is changed from alanine to threonine) to Ter DNA was estimated, compared with a value of 相似文献   

Alu-family repeat binding protein from HeLa cells which interacts with regulatory region of SV40 virus genome

Using a gel retardation assay the protein which binds selectively to the Alu-family repeat (AFR) has been identified and partially purified from HeLa cell nuclear extract. The protein (AFR-binding protein, ABP) forms multiple discrete complexes with AFR even in the presence of 200 to 2000-fold excess of non-specific (E. coli) DNA. The most stable complex has a relative mobility in 4% polyacrylamide gel (as compared to the free Alu-fragment) of 0.54. Heterogeneity of protein-DNA bands seen in the polyacrylamide gel suggests that ABP is able to form multimeric complexes with AFR. Competition experiments show that ABP do not interact with the RNA polymerase III promoter and with the TGGCA-sequence, but a high affinity binding site for ABP was found within a 660 bp restriction fragment containing the SV40 virus promoter and replication origin.     

Structure of eukaryotic DNA binding sites for steroid hormone-apolipoprotein A-I complexes

High affinity for DNA and synthetic oligonucleotides was detected for apolipoprotein A-I (ApoA-I) by affinity chromatography, affinity modification, and enzymatic analysis. Competitive inhibition and Southern hybridization showed that the tetrahydrocortisol (THC)-ApoA-I complex specifically bound to high-molecular-weight DNA in regions containing GCC/CGG sequences. The CC(GCC)₃ · GG(CGG)₃ duplex was found to be sensitive to nuclease S1 under the action of the THC-ApoA-I complex. The eukaryotic DNA binding sites for steroid (THC, androsterone)-ApoA-I complexes were found to be involved in the initiation of DNA copying in vitro.     

Role of the amino-terminal region of the DnaA protein in opening of the duplex DNA at the oriC region

We report in this paper that the amino acid residues Ile-26 and Leu-40 of the DnaA protein are essential for the DNA replication activity in vitro. Lines of evidence to support this conclusion are as follows. Variants of the DnaA protein containing either an Ile-26-Ser or Leu-40-Ser replacement were unable to support oriC DNA replication in vitro. Though the mutant DnaA proteins retained the capability to bind oriC DNA, they were unable to open the duplex DNA at oriC. Based on these and other results, we conclude that the N-terminal region of the DnaA protein is involved in the oligomerization of this protein, an essential step for the duplex opening activity at oriC.     

Identification of a second DNA binding site in human DNA methyltransferase 3A by substrate inhibition and domain deletion

The human DNA methyltransferase 3A (DNMT3A) is essential for establishing DNA methylation patterns. Knowing the key factors involved in the regulation of mammalian DNA methylation is critical to furthering understanding of embryonic development and designing therapeutic approaches targeting epigenetic mechanisms. We observe substrate inhibition for the full length DNMT3A but not for its isolated catalytic domain, demonstrating that DNMT3A has a second binding site for DNA. Deletion of recognized domains of DNMT3A reveals that the conserved PWWP domain is necessary for substrate inhibition and forms at least part of the allosteric DNA binding site. The PWWP domain is demonstrated here to bind DNA in a cooperative manner with μM affinity. No clear sequence preference was observed, similar to previous observations with the isolated PWWP domain of Dnmt3b but with one order of magnitude weaker affinity. Potential roles for a low affinity, low specificity second DNA binding site are discussed.     

Proteomic analysis of nuclear factors binding to an intronic enhancer in the myelin proteolipid protein gene

The myelin proteolipid protein gene ( Plp1 ) encodes the most abundant protein found in CNS myelin, accounting for nearly one-half of the total protein. Its expression in oligodendrocytes is developmentally regulated – peaking during the active myelination period of CNS development. Previously, we have identified a novel enhancer (designated ASE) in intron 1 DNA that appears to be important in mediating the surge of Plp1 gene activity during the active myelination period. Evidence suggests that the ASE participates in the formation of a specialized multi-protein/DNA complex called an enhanceosome. The current study describes an optimized, five-step, DNA affinity chromatography purification procedure to purify nuclear proteins from mouse brain that bind to the 85-bp ASE sequence, specifically. Electrophoretic mobility shift assay analysis demonstrated that specific DNA-binding activity was retained throughout the purification procedure, resulting in concomitant enrichment of nucleoprotein complexes. Identification of the purported regulatory factors was achieved through mass spectrometry analysis and included over 20 sequence-specific DNA-binding proteins. Supplementary western blot analyses to determine which of these sequence-specific factors are present in oligodendrocytes, and their developmental and regional expression in whole brain, suggest that Purα and Purβ rank highest among the candidate factors as constituents of the multi-protein complex formed on the ASE.     

Cell-line-induced mutation of the rotavirus genome alters expression of an IRF3-interacting protein

Rotavirus, a cause of severe gastroenteritis, contains a segmented double-stranded (ds)RNA genome that replicates using viral mRNAs as templates. The highly conserved 3'-consensus sequence (3'CS), UGUGACC, of the mRNAs promotes dsRNA synthesis and enhances translation. We have found that the 3'CS of the gene (g5) encoding NSP1, an antagonist of interferon signaling, undergoes rapid mutation when rhesus rotavirus (RRV) is serially passaged at high multiplicity of infection (MOI) in cells permitting high titer growth. These mutations increase the promoter activity of the g5 3'-sequence, but decrease its activity as a translation enhancer. The location of the mutations defines the minimal essential promoter for dsRNA synthesis as URN0-5CC. Under passage conditions where cell-to-cell spread of the virus is required to complete infection (low MOI), the 3'CS is retained due to the need for NSP1 to be expressed at levels sufficient to prevent establishment of the antiviral state. These data demonstrate that host cell type and propagation conditions affect the capacity of RRV to produce the virulence gene product NSP1, an important consideration in producing RRV-based vaccines.     

Biochemical and physiological properties of the DNA binding domain of AraC protein

Intact AraC protein is poorly soluble and difficult to purify, whereas its dimerization domain is the opposite. Unexpectedly, the DNA binding domain of AraC proved also to be soluble in cells when overproduced and is easily purified to homogeneity. The DNA binding affinity of the DNA binding domain for its binding site could not be measured by electrophoretic mobility shift because of its rapid association and dissociation rates, but its affinity could be measured with a fluorescence assay and was found to have a dissociation constant of 1 x 10(-8)M in 100 mM KCl. The binding of monomers of the DNA binding domain to adjacent half-sites occurs without substantial positive or negative cooperativity. A simple analysis relates the DNA binding affinities of monomers of DNA binding domain and normal dimeric AraC protein.     

A 17S multiprotein form of murine cell DNA polymerase mediates polyomavirus DNA replication in vitro

We have identified and purified a multiprotein form of DNA polymerase from the murine mammary carcinoma cell line (FM3A) using a series of centrifugation, polyethylene glycol precipitation, and ion-exchange chromatography steps. Proteins and enzymatic activities associated with this mouse cell multiprotein form of DNA polymerase include the DNA polymerases α and δ, DNA primase, proliferating cell nuclear antigen (PCNA), DNA ligase I, DNA helicase, and DNA topoisomerases I and II. The sedimentation coefficient of the multiprotein form of DNA polymerase is 17S, as determined by sucrose density gradient analysis. The integrity of the murine cell multiprotein form of DNA polymerase is maintained after treatment with detergents, salt, RNase, DNase, and after chromatography on DE52-cellulose, suggesting that the association of the proteins with one another is independent of nonspecific interaction with other cellular macromolecular components. Most importantly, we have demonstrated that this complex of proteins is fully competent to replicate polyomavirus DNA in vitro. This result implies that all of the cellular activities required for large T-antigen dependent in vitro polyomavirus DNA synthesis are present within the isolated 17S multiprotein form of the mouse cell DNA replication activities. A model is proposed to represent the mammalian Multiprotein DNA Replication Complex (MRC) based on the fractionation and chromatographic profiles of the individual proteins found to co-purify with the complex.     

Partial purification and characterization of a cellular protein that binds to the SV40 core origin of DNA replication

A monkey cell factor that interacts specifically with double- and single-stranded DNA sequences in the early domain of the simian virus 40 (SV40) core origin of replication was identified using gel-retention assays. The protein was enriched over 1200-fold using ion-exchange and affinity chromatography on single-strand DNA cellulose. Binding of protein to mutant origin DNA restriction fragments was correlated with replication activity of the mutant DNAs. Exonuclease footprint experiments on single-stranded DNA revealed prominent pause sites in the early domain of the core origin. The results suggest that this cellular protein may be involved in SV40 DNA replication.