RESUMEN
Breast carcinoma invasion is associated with prominent alterations in stromal fibroblasts. Carcinoma-associated fibroblasts (CAF) support and promote tumorigenesis, whereas normal mammary fibroblasts (NF) are thought to suppress tumor progression. Little is known about the difference in gene expression between CAF and NF or the patient-to-patient variability in gene expression. Paired CAF and NF were isolated from six primary human breast carcinoma specimens. RNA was extracted from low-passage cultures of CAF and NF and analyzed with Affymetrix Human Genome U133 Plus 2.0 arrays. The array data were examined with an empirical Bayes model and filtered according to the posterior probability of equivalent expression and fold difference in expression. Twenty-one genes (27 probe sets) were up-regulated in CAF, as compared with NF. Known functions of these genes relate to paracrine or intracellular signaling, transcriptional regulation, extracellular matrix and cell adhesion/migration. Ten genes (14 probe sets) were down-regulated in CAF, including the pluripotency transcription factor KLF4. Quantitative RT-PCR analysis of 10 genes validated the array results. Immunohistochemical staining for three gene products confirmed stromal expression in terms of location and relative quantity. Surprisingly, the variability of gene expression was slightly higher in NF than in CAF, suggesting inter-individual heterogeneity of normal stroma.
Asunto(s)
Neoplasias de la Mama/genética , Mama/fisiología , Fibroblastos/fisiología , Regulación Neoplásica de la Expresión Génica , Regulación de la Expresión Génica , Células del Estroma/fisiología , Teorema de Bayes , Mama/citología , Proteínas CCN de Señalización Intercelular , Femenino , Fibroblastos/citología , Fibroblastos/patología , Variación Genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Embarazo , Probabilidad , Proteínas Proto-Oncogénicas/genética , Transducción de Señal , Células del Estroma/citología , Transcripción GenéticaAsunto(s)
Antígenos de Histocompatibilidad Clase I/inmunología , Macaca mulatta/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Linfocitos T/inmunología , Proteínas Reguladoras y Accesorias Virales/inmunología , Animales , Pruebas Inmunológicas de Citotoxicidad , Epítopos , Exones , Frecuencia de los Genes , Antígenos de Histocompatibilidad Clase I/genética , Intrones , Reacción en Cadena de la PolimerasaRESUMEN
Here we describe a nucleotide insertion in intron 2 of two rhesus major histocompatibility complex (MHC) class I alleles, Mamu-A*05 and Mamu-A*07. This resulted in an intron 2 that was nearly twice the length of any other intron 2 of primate MHC class I genes sequenced to date. This insertion was most similar (93% identity) to the beginning of intron 3 of HLA-A alleles. It was also similar to intron 3 of several human MHC class I pseudogenes and MHC class I pseudogenes from cotton top tamarin and cat. The finding of this insertion in two rhesus MHC class I genes is surprising given the uniformity in length and sequence of introns of functional HLA-A, -B and -C genes.
Asunto(s)
Alelos , Antígenos de Histocompatibilidad Clase I/genética , Intrones , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario , Humanos , Macaca mulatta , Datos de Secuencia Molecular , Mutagénesis Insercional , Homología de Secuencia de Ácido NucleicoRESUMEN
The RecA, UmuC, and UmuD' proteins are essential for error-prone, replicative bypass of DNA lesions. Normally, RecA protein mediates homologous pairing of DNA. We show that purified Umu(D')2C blocks this recombination function. Biosensor measurements establish that the mutagenic complex binds to the RecA nucleoprotein filament with a stoichiometry of one Umu(D')2C complex for every two RecA monomers. Furthermore, Umu(D')2C competitively inhibits LexA repressor cleavage but not ATPase activity, implying that Umu(D')2C binds in or proximal to the helical groove of the RecA nucleoprotein filament. This binding reduces joint molecule formation and even more severely impedes DNA heteroduplex formation by RecA protein, ultimately blocking all DNA pairing activity and thereby abridging participation in recombination function. Thus, Umu(D')2C restricts the activities of the RecA nucleoprotein filament and presumably, in this manner, recruits it for mutagenic repair function. This modulation by Umu(D')2C is envisioned as a key event in the transition from a normal mode of genomic maintenance by "error-free" recombinational repair, to one of "error-prone" DNA replication.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Mutágenos/metabolismo , Rec A Recombinasas/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN , Mutagénesis , Unión ProteicaRESUMEN
Essential to the two distinct cellular events of genetic recombination and SOS induction in Escherichia coli, RecA protein promotes the homologous pairing and exchange of DNA strands and the proteolytic cleavage of the LexA repressor, respectively. Since both of these activities require single-stranded DNA (ssDNA) and ATP, the inter-relationship between these reactions was investigated and found to display many parallels. The extent of active complex formed between RecA protein and M13 ssDNA, as measured by both ATP hydrolysis and LexA proteolysis, is stimulated in a similar manner by either a reduction in magnesium ion concentration or the presence of single-stranded DNA binding (SSB) protein. However, unexpectedly, SSB protein inhibits both LexA proteolysis and ATP hydrolysis (in assays containing repressor) at concentrations of RecA protein that are substoichiometric to the ssDNA, arguing that LexA repressor affects the competition between RecA and SSB proteins for limited ssDNA binding sites. Additionally, attenuation of LexA repressor cleavage in the presence of double-stranded DNA or by an excess of ssDNA suggests that interaction of the RecA nucleoprotein filament with either LexA repressor or a secondary DNA molecule is mutually exclusive. The significance of these results is discussed in the context of both the regulation of inducible responses to DNA damage, and the competitive relationship between the processes of SOS induction and genetic recombination.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Rec A Recombinasas/metabolismo , Serina Endopeptidasas/metabolismo , Adenosina Trifosfato/metabolismo , Desoxirribonucleoproteínas/química , Hidrólisis , Sustancias Macromoleculares , Magnesio/metabolismo , Recombinación Genética , Respuesta SOS en GenéticaRESUMEN
The Escherichia coli RecA protein is involved in SOS induction, DNA repair, and homologous recombination. In vitro, RecA protein serves as a co-protease to cleave LexA repressor, the repressor of the SOS regulon; in addition, RecA protein promotes homologous pairing and DNA strand exchange, steps important to homologous recombination and DNA repair. To determine if these two functions of RecA protein are competing or parallel, the effect of uncleavable LexA S119A repressor on RecA protein-dependent activities was examined. LexA S119A repressor inhibits both the single-stranded DNA (ssDNA)-dependent ATP hydrolysis and DNA strand exchange activities of RecA protein. As for wild-type LexA repressor (Rehrauer, W. M., Lavery, P. E., Palmer, E. L., Singh, R. N., and Kowalczykowski, S. C. (1996) J. Biol. Chem. 271, 23865-23873), inhibition of ATP hydrolysis is dependent upon the presence of E. coli single-stranded DNA binding (SSB) protein, arguing that LexA repressor affects the competition between RecA protein and SSB protein for ssDNA binding sites. In contrast, inhibition of DNA strand exchange activity is SSB protein-independent, suggesting that LexA S119A repressor blocks a site required for DNA strand exchange. These results imply that there is a common site on the RecA protein filament for secondary DNA and LexA repressor binding and raise the possibility that the recombination and co-protease activities of the RecA protein filament are competitive.
Asunto(s)
Proteínas Bacterianas/fisiología , ADN de Cadena Simple/metabolismo , Escherichia coli/genética , Rec A Recombinasas/metabolismo , Recombinación Genética , Respuesta SOS en Genética , Serina Endopeptidasas/fisiología , Adenosina Trifosfatasas/metabolismo , Unión Competitiva , Hidrólisis , Sustancias MacromolecularesRESUMEN
Photochemical cross-linking has been used to identify residues in the Escherichia coli RecA protein that are proximal to and may directly mediate binding of DNA. Ultraviolet irradiation promotes specific and efficient cross-linking of the RecA protein to poly(deoxythymidylic) acid. Cross-linked peptides remaining covalently attached to the polynucleotide following proteolytic digestion with trypsin correspond to amino acids 61-72, 178-183, and 233-243 of the RecA protein primary sequence. Their location and surface accessibility in the crystal structure, along with the behavior of various recA mutants, support the assignment of the cross-linked regions to the DNA binding site(s) of the RecA protein. Functional overlap of amino acids 61-72 with an element of the ATP binding site suggests a structural mechanism by which nucleotide cofactors allosterically affect the RecA nucleoprotein filament.
Asunto(s)
ADN/metabolismo , Rec A Recombinasas/química , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Regulación Alostérica , Secuencia de Aminoácidos , Sitios de Unión , Cristalización , Datos de Secuencia Molecular , Mutación , Rec A Recombinasas/metabolismoRESUMEN
Homologous recombination is a fundamental biological process. Biochemical understanding of this process is most advanced for Escherichia coli. At least 25 gene products are involved in promoting genetic exchange. At present, this includes the RecA, RecBCD (exonuclease V), RecE (exonuclease VIII), RecF, RecG, RecJ, RecN, RecOR, RecQ, RecT, RuvAB, RuvC, SbcCD, and SSB proteins, as well as DNA polymerase I, DNA gyrase, DNA topoisomerase I, DNA ligase, and DNA helicases. The activities displayed by these enzymes include homologous DNA pairing and strand exchange, helicase, branch migration, Holliday junction binding and cleavage, nuclease, ATPase, topoisomerase, DNA binding, ATP binding, polymerase, and ligase, and, collectively, they define biochemical events that are essential for efficient recombination. In addition to these needed proteins, a cis-acting recombination hot spot known as Chi (chi: 5'-GCTGGTGG-3') plays a crucial regulatory function. The biochemical steps that comprise homologous recombination can be formally divided into four parts: (i) processing of DNA molecules into suitable recombination substrates, (ii) homologous pairing of the DNA partners and the exchange of DNA strands, (iii) extension of the nascent DNA heteroduplex; and (iv) resolution of the resulting crossover structure. This review focuses on the biochemical mechanisms underlying these steps, with particular emphases on the activities of the proteins involved and on the integration of these activities into likely biochemical pathways for recombination.
Asunto(s)
Escherichia coli/genética , Exodesoxirribonucleasas/metabolismo , Modelos Genéticos , Rec A Recombinasas/metabolismo , Recombinación Genética/genética , Escherichia coli/metabolismo , Exodesoxirribonucleasa V , Rec A Recombinasas/genéticaRESUMEN
The hydrolysis of the nucleoside triphosphates, such as ATP or GTP, plays a central role in a variety of biochemical processes; but, in most cases, the specific mechanism of energy transduction is unclear. DNA strand exchange promoted by the Escherichia coli recA protein is normally associated with ATP hydrolysis. However, we advanced the idea that the observed ATP hydrolysis is not obligatorily linked to the exchange of DNA strands (Menetski, J. P., Bear, D. G., and Kowalczykowski, S. C. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 21-25); instead, ATP binding resulting in an allosteric transition to an active form of the recA protein is sufficient. In this paper, we extend this conclusion by introducing a mutation within a highly conserved region of the recA protein that, on the basis of sequence similarity, is proposed to interact with the pyrophosphate moiety of a bound NTP molecule. The conservative substitution of an arginine for the invariant lysine at position 72 reduces NTP hydrolysis by approximately 600-850-fold. This mutation does not significantly alter the capacity of the mutant recA (K72R) protein either to bind nucleotide cofactors and single-stranded DNA or to respond allosterically to nucleotide cofactor binding. Despite the dramatic attenuation in NTP hydrolysis, the recA (K72R) protein retains the ability to promote homologous pairing and extensive exchange of DNA strands (up to 1.5 kilobase pairs). These results both identify a component of the catalytic domain for NTP hydrolysis and demonstrate that the recA protein-promoted pairing and exchange of DNA strands mechanistically require the allosteric transition induced by NTP cofactor binding, but not the energy educed from NTP hydrolysis.