RESUMO
Telomere repeat sequences cap the ends of eucaryotic chromosomes and help stabilize them. At interstitial sites, however, they may destabilize chromosomes, as suggested by cytogenetic studies in mammalian cells that correlate interstitial telomere sequence with sites of spontaneous and radiation-induced chromosome rearrangements. In no instance is the length, purity, or orientation of the telomere repeats at these potentially destabilizing interstitial sites known. To determine the effects of a defined interstitial telomere sequence on chromosome instability, as well as other aspects of DNA metabolism, we deposited 800 bp of the functional vertebrate telomere repeat, TTAGGG, in two orientations in the second intron of the adenosine phosphoribosyltransferase (APRT) gene in Chinese hamster ovary cells. In one orientation, the deposited telomere sequence did not interfere with expression of the APRT gene, whereas in the other it reduced mRNA levels slightly. The telomere sequence did not induce chromosome truncation and the seeding of a new telomere at a frequency above the limits of detection. Similarly, the telomere sequence did not alter the rate or distribution of homologous recombination events. The interstitial telomere repeat sequence in both orientations, however, dramatically increased gene rearrangements some 30-fold. Analysis of individual rearrangements confirmed the involvement of the telomere sequence. These studies define the telomere repeat sequence as a destabilizing element in the interior of chromosomes in mammalian cells.
Assuntos
Adenina Fosforribosiltransferase/genética , Fragilidade Cromossômica/genética , Mutagênese Insercional/genética , Sequências Repetitivas de Ácido Nucleico/genética , Telômero/genética , Animais , Sequência de Bases , Southern Blotting , Células CHO , Deleção Cromossômica , Cricetinae , Marcação de Genes , Íntrons/genética , Dados de Sequência Molecular , Fenótipo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Recombinação Genética/genética , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Timidina Quinase/genéticaRESUMO
Spontaneous recombination between direct repeats at the adenine phosphoribosyltransferase (APRT) locus in ERCC1-deficient cells generates a high frequency of rearrangements that are dependent on the process of homologous recombination, suggesting that rearrangements are formed by misprocessing of recombination intermediates. Given the specificity of the structure-specific Ercc1/Xpf endonuclease, two potential recombination intermediates are substrates for misprocessing in ERCC1(-) cells: heteroduplex loops and heteroduplex intermediates with non-homologous 3' tails. To investigate the roles of each, we constructed repeats that would yield no heteroduplex loops during spontaneous recombination or that would yield two non-homologous 3' tails after treatment with the rare-cutting endonuclease I-SCE:I. Our results indicate that misprocessing of heteroduplex loops is not the major source of recombination-dependent rearrangements in ERCC1-deficient cells. Our results also suggest that the Ercc1/Xpf endonuclease is required for efficient removal of non-homologous 3' tails, like its Rad1/Rad10 counterpart in yeast. Thus, it is likely that misprocessing of non-homologous 3' tails is the primary source of recombination-dependent rearrangements in mammalian cells. We also find an unexpected effect of ERCC1 deficiency on I-SCE:I-stimulated rearrangements, which are not dependent on homologous recombination, suggesting that the ERCC1 gene product may play a role in generating the rearrangements that arise after I-SCE:I-induced double-strand breaks.
Assuntos
Reparo do DNA/genética , Proteínas de Ligação a DNA , DNA/química , DNA/metabolismo , Endonucleases , Proteínas/metabolismo , Recombinação Genética/genética , Adenina Fosforribosiltransferase/genética , Animais , Southern Blotting , Linhagem Celular , Troca Genética/genética , DNA/genética , Dano ao DNA/genética , Deleção de Genes , Conformação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/química , Ácidos Nucleicos Heteroduplexes/genética , Ácidos Nucleicos Heteroduplexes/metabolismo , Proteínas/genética , Sequências Repetitivas de Ácido Nucleico/genética , Especificidade por Substrato , TransfecçãoRESUMO
Nucleotide excision repair proteins have been implicated in genetic recombination by experiments in Saccharomyces cerevisiae and Drosophila melanogaster, but their role, if any, in mammalian cells is undefined. To investigate the role of the nucleotide excision repair gene ERCC1, the hamster homologue to the S. cerevisiae RADIO gene, we disabled the gene by targeted knockout. Partial tandem duplications of the adenine phosphoribosyltransferase (APRT) gene then were constructed at the endogenous APRT locus in ERCC1- and ERCC1+ cells. To detect the full spectrum of gene-altering events, we used a loss-of-function assay in which the parental APRT+ tandem duplication could give rise to APRT- cells by homologous recombination, gene rearrangement, or point mutation. Measurement of rates and analysis of individual APRT- products indicated that gene rearrangements (principally deletions) were increased at least 50-fold, whereas homologous recombination was affected little. The formation of deletions is not caused by a general effect of the ERCC1 deficiency on gene stability, because ERCC1- cell lines with a single wild-type copy of the APRT gene yielded no increase in deletions. Thus, deletion formation is dependent on the tandem duplication, and presumably the process of homologous recombination. Recombination-dependent deletion formation in ERCC1- cells is supported by a significant decrease in a particular class of crossover products that are thought to arise by repair of a heteroduplex intermediate in recombination. We suggest that the ERCC1 gene product in mammalian cells is involved in the processing of heteroduplex intermediates in recombination and that the misprocessed intermediates in ERCC1- cells are repaired by illegitimate recombination.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA , Endonucleases , Proteínas/genética , Recombinação Genética , Deleção de Sequência , Adenina Fosforribosiltransferase/genética , Animais , Linhagem Celular , Cricetinae , Timidina Quinase/genéticaRESUMO
The retinoblastoma protein (p110RB) interacts with many cellular proteins in complexes potentially important for its growth-suppressing function. We have developed and used an improved version of the yeast two-hybrid system to isolate human cDNAs encoding proteins able to bind p110RB. One clone encodes a novel type 1 protein phosphatase catalytic subunit (PP-1 alpha 2), which differs from the originally defined PP-1 alpha by an amino-terminal 11-amino-acid insert. In vitro-binding assays demonstrated that PP-1 alpha isoforms preferentially bind the hypophosphorylated form of p110RB. Moreover, similar p110RB sequences are required for binding PP-1 alpha 2 and SV40 large T antigen. Cell cycle synchrony experiments revealed that this association occurs from mitosis to early G1. The implications of these findings on the regulation of both proteins are discussed.