Assuntos
Atenção à Saúde/organização & administração , Saúde Global , Internacionalidade , Criança , Serviços de Saúde da Criança/organização & administração , Dieta , Serviços de Planejamento Familiar/organização & administração , Infecções por HIV/tratamento farmacológico , Infecções por HIV/prevenção & controle , Humanos , Malária/tratamento farmacológico , Malária/prevenção & controle , Serviços de Saúde Materna/organização & administração , North Carolina , Tuberculose/tratamento farmacológico , Tuberculose/prevenção & controleRESUMO
Among several different types of repetitive sequences found in the human genome, this study has examined the telomeric repeat, necessary for the protection of chromosome termini, and the disease-associated triplet repeat (CTG).(CAG)n. Evidence suggests that replication of both types of repeats is problematic and that a contributing factor is the repetitive nature of the DNA itself. Here we have used electron microscopy to investigate DNA structures formed at replication forks on large model DNAs containing these repeat sequences, in an attempt to elucidate the contributory effect that these repetitive DNAs may have on their replication. Visualization of the DNA revealed that there is a high propensity for a paused replication fork to spontaneously regress when moving through repetitive DNAs, and that this results in a four-way chickenfoot intermediate that could present a significant block to replication in vivo, possibly leading to unwanted recombination events, amplifications or deletions.
Assuntos
Replicação do DNA , DNA/ultraestrutura , Sequências Repetitivas de Ácido Nucleico , Telômero/química , DNA/química , Humanos , Moldes Genéticos , Repetições de TrinucleotídeosRESUMO
The replication of long tracts of telomeric repeats may require specific factors to avoid fork regression (Fouché, N., Ozgür, S., Roy, D., and Griffith, J. (2006) Nucleic Acids Res., in press). Here we show that TRF2 binds to model replication forks and four-way junctions in vitro in a structure-specific but sequence-independent manner. A synthetic peptide encompassing the TRF2 basic domain also binds to DNA four-way junctions, whereas the TRF2 truncation mutant (TRF2(DeltaB)) and a mutant basic domain peptide do not. In the absence of the basic domain, the ability of TRF2 to localize to model telomere ends and facilitate t-loop formation in vitro is diminished. We propose that TRF2 plays a key role during telomere replication in binding chickenfoot intermediates of telomere replication fork regression. Junction-specific binding would also allow TRF2 to stabilize a strand invasion structure that is thought to exist at the strand invasion site of the t-loop.
Assuntos
DNA/química , DNA/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Semelhantes à Proteína de Ligação a TATA-Box/química , Proteínas Semelhantes à Proteína de Ligação a TATA-Box/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , DNA/genética , DNA/ultraestrutura , Sondas de DNA/genética , DNA Cruciforme/química , DNA Cruciforme/genética , DNA Cruciforme/metabolismo , DNA Cruciforme/ultraestrutura , Humanos , Técnicas In Vitro , Microscopia Eletrônica , Dados de Sequência Molecular , Proteínas Nucleares/genética , Conformação de Ácido Nucleico , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sequências Repetitivas de Ácido Nucleico , Deleção de Sequência , Proteínas Semelhantes à Proteína de Ligação a TATA-Box/genética , Proteína 1 de Ligação a Repetições Teloméricas/química , Proteína 1 de Ligação a Repetições Teloméricas/genética , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Proteína 2 de Ligação a Repetições TeloméricasRESUMO
Binding of the telomerase ribonucleoprotein from the ciliate Euplotes aediculatus to telomeric DNA in vitro has been examined by electron microscopy (EM). Visualization of the structures that formed revealed a globular protein complex that localized to the DNA end containing the E. aediculatus telomere consensus 3'-single-strand T(4)G(4)T(4)G(4)T(4)G(2) overhang. Gel filtration confirmed that purified E. aediculatus telomerase is an active dimer in solution, and comparison of the size of the DNA-associated complex with apoferritin suggests that E. aediculatus telomerase binds to a single telomeric 3'-end as a dimer. Up to 43% of the telomerase-DNA complexes appeared by EM to involve tetramers or larger multimers of telomerase in association with two or more DNA ends. These data provide the first direct evidence that telomerase is a functional dimer and suggest that two telomerase ribonucleoprotein particles cooperate to elongate each Euplotes telomere in vivo.