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EXO1 resection at G-quadruplex structures facilitates resolution and replication.

ABSTRACT: G-quadruplexes represent unique roadblocks to DNA replication, which tends to stall at these secondary structures. Although G-quadruplexes can be found throughout the genome, telomeres, due to their G-richness, are particularly predisposed to forming these structures and thus represent difficult-to-replicate regions. Here, we demonstrate that exonuclease 1 (EXO1) plays a key role in the resolution of, and replication through, telomeric G-quadruplexes. When replication forks encounter G-quadruplexes, EXO1 resects the nascent DNA proximal to these structures to facilitate fork progression and faithful replication. In the absence of EXO1, forks accumulate at stabilized G-quadruplexes and ultimately collapse. These collapsed forks are preferentially repaired via error-prone end joining as depletion of EXO1 diverts repair away from error-free homology-dependent repair. Such aberrant repair leads to increased genomic instability, which is exacerbated at chromosome termini in the form of dysfunction and telomere loss.

SUBMITTER: Stroik S

PROVIDER: S-EPMC7229832 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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EXO1 resection at G-quadruplex structures facilitates resolution and replication.

Stroik Susanna S Kurtz Kevin K Lin Kevin K Karachenets Sergey S Myers Chad L CL Bielinsky Anja-Katrin AK Hendrickson Eric A EA

Nucleic acids research 20200501 9

G-quadruplexes represent unique roadblocks to DNA replication, which tends to stall at these secondary structures. Although G-quadruplexes can be found throughout the genome, telomeres, due to their G-richness, are particularly predisposed to forming these structures and thus represent difficult-to-replicate regions. Here, we demonstrate that exonuclease 1 (EXO1) plays a key role in the resolution of, and replication through, telomeric G-quadruplexes. When replication forks encounter G-quadruple ...[more]

PMID: 32232411

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Project description:Replication protein A (RPA) is known to interact with guanine- (G-) rich sequences that adopt G-quadruplex (GQ) structures. Most studies reported in the literature were performed on GQ formed by homogeneous sequences, such as the human telomeric repeat, and RPA's ability to unfold GQ structures of differing stability is not known. We compared the thermal stability of three potential GQ-forming DNA sequences (PQSs) to their stability against RPA-mediated unfolding using single-molecule fluorescence resonance energy transfer (FRET) and bulk biophysical and biochemical experiments. One of these sequences is the human telomeric repeat and the other two, located in the promoter region of tyrosine hydroxylase gene, are highly heterogeneous sequences that better represent PQSs in the genome. The three GQ constructs have thermal stabilities that differ significantly. Our measurements showed that the most thermally stable structure (Tm = 86 °C) was also the most stable against RPA-mediated unfolding, although the least thermally stable structure (Tm = 69 °C) had at least an order-of-magnitude higher stability against RPA-mediated unfolding than the structure with intermediate thermal stability (Tm = 78 °C). The significance of this observation becomes more evident when considered within the context of the cellular environment where protein-DNA interactions can be an important determinant of GQ viability. Considering these results, we conclude that thermal stability is not necessarily an adequate criterion for predicting the physiological viability of GQ structures. Finally, we measured the time it takes for an RPA molecule to unfold a GQ from a fully folded to a fully unfolded conformation using a single-molecule stopped-flow method. All three GQ structures were unfolded within Δt ≈ 0.30 ± 0.10 s, a surprising result considering that the unfolding time does not correlate with thermal stability or stability against RPA-mediated unfolding. These results suggest that the limiting step in G-quadruplex unfolding by RPA is simply the accessibility of the structure to the RPA protein.

Project description:DNA secondary structures are important for fundamental genome functions such as transcription and replication1. The G-quadruplex (G4) structural motif has been linked to gene regulation2,3 and genome instability4,5 and may be important to cancer development and other diseases6-8. Recently, ~700,000 discrete G4s have been observed in naked human single-stranded genomic DNA using G4-seq, a high-throughput sequencing technique that detects structural features in vitro.9 It is of vital importance to investigate G4 structures within an endogenous chromatin context, which until now remained elusive10,11. Herein, we address this via the development of G4 ChIP-seq, an antibody-based G4 chromatin immunoprecipitation and high-throughput sequencing approach. We identified ~10,000 endogenous G4 structures and show that G4s are predominantly seen in regulatory, nucleosome-depleted, chromatin regions. G4s were enriched in the promoters and 5âUTR regions of highly transcribed genes, particularly in genes related to cancer and in somatic copy number amplifications, such as MYC. Reorganization of the chromatin landscape using a histone deacetylase inhibitor, resulted in de novo G4 formation in new and more prominent regulatory, nucleosome-depleted regions associated with increased transcriptional output. Our findings suggest a striking relationship between promoter nucleosome-depleted regions, G4 formation and elevated transcriptional activity. Comparison between normal human epidermal keratinocytes and their immortalized counterparts revealed a ï¾7-fold greater G4 abundance in immortalized cells, of which 80 % were found in regulatory, nucleosome-depleted regions common to both cell types. Consequently, cells exhibiting more G4s displayed significantly increased transcriptional output and were more sensitive to growth inhibition by a small molecule G4 ligand. Overall, our results provide new mechanistic insights into where and when DNA adopts G4 structure in vivo. Our findings show for the first time that regulatory, nucleosome-depleted chromatin and transcriptional states predominantly shape the endogenous G4 DNA landscape. Two cell lines, treated with entinostat or untreated, analyzed to detect gene expression differences, presence of G-Qudruplexes and chromatin state. Each combination of conditions replicated in duplicates or triplicates.

Dataset Information

EXO1 resection at G-quadruplex structures facilitates resolution and replication.

Publications

EXO1 resection at G-quadruplex structures facilitates resolution and replication.

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