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1.
bioRxiv ; 2024 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-38559215

RESUMO

Putative G-quadruplex forming sequences (PQS) have been identified in promoter sequences of prominent genes that are implicated among others in cancer and neurological disorders. We explored mechanistic aspects of CRISPR-dCas9-mediated gene expression regulation, which is transient and sequence specific unlike alternative approaches that lack such specificity or create permanent mutations, using the PQS in tyrosine hydroxylase (TH) and c-Myc promoters as model systems. We performed in vitro ensemble and single molecule investigations to study whether G-quadruplex (GQ) structures or dCas9 impede T7 RNA polymerase (RNAP) elongation process and whether orientation of these factors is significant. Our results demonstrate that dCas9 is more likely to block RNAP progression when the non-template strand is targeted. While the GQ in TH promoter was effectively destabilized when the dCas9 target site partially overlapped with the PQS, the c-Myc GQ remained folded and stalled RNAP elongation. We also determined that a minimum separation between the transcription start site and the dCas9 target site is required for effective stalling of RNAP by dCas9. Our study provides significant insights about the factors that impact dCas9-mediated transcription regulation when dCas9 targets the vicinity of sequences that form secondary structures and provides practical guidelines for designing guide RNA sequences.

2.
J Mol Biol ; 436(1): 168205, 2024 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-37481156

RESUMO

Telomeres and their single stranded overhangs gradually shorten with successive cell divisions, as part of the natural aging process, but can be elongated by telomerase, a nucleoprotein complex which is activated in the majority of cancers. This prominent implication in cancer and aging has made the repetitive telomeric sequences (TTAGGG repeats) and the G-quadruplex structures that form in their overhangs the focus of intense research in the past several decades. However, until recently most in vitro efforts to understand the structure, stability, dynamics, and interactions of telomeric overhangs had been focused on short sequences that are not representative of longer sequences encountered in a physiological setting. In this review, we will provide a broad perspective about telomeres and associated factors, and introduce the agents and structural characteristics involved in organizing, maintaining, and protecting telomeric DNA. We will also present a summary of recent research performed on long telomeric sequences, nominally defined as those that can form two or more tandem G-quadruplexes, i.e., which contain eight or more TTAGGG repeats. Results of experimental studies using a broad array of experimental tools, in addition to recent computational efforts will be discussed, particularly in terms of their implications for the stability, folding topology, and compactness of the tandem G-quadruplexes that form in long telomeric overhangs.


Assuntos
DNA , Quadruplex G , Telomerase , Telômero , DNA/genética , DNA/química , Sequências Repetitivas de Ácido Nucleico/genética , Telomerase/genética , Telômero/genética , Telômero/metabolismo , Humanos , Animais
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