Cleavage-defective Topoisomerase I mutants sharply increase G-quadruplex-associated genomic instability.

Topoisomerase 1 (Top1) removes transcription-associated helical stress to suppress G4-formation and its induced recombination at genomic loci containing guanine-run containing sequences. Interestingly, Top1 binds tightly to G4 structures, and its inhibition or depletion can cause elevated instability at these genomic loci. Top1 is targeted by the widely used anti-cancer chemotherapeutic camptothecin (CPT) and its derivatives, which stabilize Top1 covalently attached on a DNA nick and prevent the re-ligation step. Here we investigated how CPT-resistance conferring Top1 mutants, which emerge in cancer patients and cells treated with CPT, affect G4-induced genomic instability in S. cerevisiae. We found that Top1 mutants form stable complexes with G4 DNA and that expression of Top1 cleavage-defective mutants but not a DNA-binding-defective mutant lead to significantly elevated instability at a G4-forming genomic locus. Elevated recombination rates were partly suppressed by their proteolytic removal by SPRTN homolog Wss1 SUMO-dependent metalloprotease in vivo. Furthermore, interaction between G4-DNA binding protein Nsr1, a homolog to clinically-relevant human nucleolin, and Top1 mutants lead to a synergistic increase in G4-associated recombination. These results in the yeast system are strengthened by our cancer genome data analyses showing that functionally detrimental mutations in Top1 correlate with an enrichment of mutations at G4 motifs. Our collective experimental and computational findings point to cooperative binding of Top1 cleavage-defective mutants and Nsr1 as promoting DNA replication blockage and exacerbating genomic instability at G4-motifs, thus complicating patient treatment.

The activity of yeast Apn2 AP endonuclease at uracil-derived AP sites is dependent on the major carbon source.

Yeast Apn2 is an AP endonuclease and DNA 3'-diesterase that belongs to the Exo III family with homology to the E. coli exonuclease III, Schizosaccharomyces pombe eth1, and human AP endonucleases APEX1 and APEX2. In the absence of Apn1, the major AP endonuclease in yeast, Apn2 can cleave the DNA backbone at an AP lesion initiating the base excision repair pathway. To study the role and relative contribution of Apn2, we took advantage of a reporter system that was previously used to delineate how uracil-derived AP sites are repaired. At this reporter, disruption of the Apn1-initiated base excision repair pathway led to a significant elevation of A:T to C:G transversions. Here we show that such highly elevated A:T to C:G transversion mutations associated with uracil residues in DNA are abolished when apn1∆ yeast cells are grown in glucose as the primary carbon source. We also show that the disruption of Apn2, either by the complete gene deletion or by the mutation of a catalytic residue, results in a similarly reduced rate of the uracil-associated mutations. Overall, our results indicate that Apn2 activity is regulated by the glucose repression pathway in yeast.

Yeast Nucleolin Nsr1 Impedes Replication and Elevates Genome Instability at an Actively Transcribed Guanine-Rich G4 DNA-Forming Sequence.

A significant increase in genome instability is associated with the conformational shift of a guanine-run-containing DNA strand into the four-stranded G-quadruplex (G4) DNA. The mechanism underlying the recombination and genome rearrangements following the formation of G4 DNA in vivo has been difficult to elucidate but has become better clarified by the identification and functional characterization of several key G4 DNA-binding proteins. Mammalian nucleolin (NCL) is a highly specific G4 DNA-binding protein with a well-defined role in the transcriptional regulation of genes with associated G4 DNA-forming sequence motifs at their promoters. The consequence of the in vivo interaction between G4 DNA and nucleolin in respect to the genome instability has not been previously investigated. We show here that the yeast nucleolin Nsr1 is enriched at a G4 DNA-forming sequence in vivo and is a major factor in inducing the genome instability associated with the cotranscriptionally formed G4 DNA in the yeast genome. We also show that Nsr1 results in impeding replication past such a G4 DNA-forming sequence. The G4-associated genome instability and the G4 DNA-binding in vivo require the arginine-glycine-glycine (RGG) repeats located at the C-terminus of the Nsr1 protein. Nsr1 with the deletion of RGG domain supports normal cell growth and is sufficient for its pre-rRNA processing function. However, the truncation of the RGG domain of Nsr1 significantly weakens its interaction with G4 DNA in vivo and restores unhindered replication, overall resulting in a sharp reduction in the genome instability associated with a guanine-rich G4 DNA-forming sequence. Our data suggest that the interaction between Nsr1 with the intact RGG repeats and G4 DNA impairs genome stability by precluding the access of G4-resolving proteins and impeding replication.

Characterization of long G4-rich enhancer-associated genomic regions engaging in a novel loop:loop 'G4 Kissing' interaction.

Mammalian antibody switch regions (∼1500 bp) are composed of a series of closely neighboring G4-capable sequences. Whereas numerous structural and genome-wide analyses of roles for minimal G4s in transcriptional regulation have been reported, Long G4-capable regions (LG4s)-like those at antibody switch regions-remain virtually unexplored. Using a novel computational approach we have identified 301 LG4s in the human genome and find LG4s prone to mutation and significantly associated with chromosomal rearrangements in malignancy. Strikingly, 217 LG4s overlap annotated enhancers, and we find the promoters regulated by these enhancers markedly enriched in G4-capable sequences suggesting G4s facilitate promoter-enhancer interactions. Finally, and much to our surprise, we also find single-stranded loops of minimal G4s within individual LG4 loci are frequently highly complementary to one another with 178 LG4 loci averaging >35 internal loop:loop complements of >8 bp. As such, we hypothesized (then experimentally confirmed) that G4 loops within individual LG4 loci directly basepair with one another (similar to characterized stem-loop kissing interactions) forming a hitherto undescribed, higher-order, G4-based secondary structure we term a 'G4 Kiss or G4K'. In conclusion, LG4s adopt novel, higher-order, composite G4 structures directly contributing to the inherent instability, regulatory capacity, and maintenance of these conspicuous genomic regions.

The Functional Consequences of Eukaryotic Topoisomerase 1 Interaction with G-Quadruplex DNA.

Topoisomerase I in eukaryotic cells is an important regulator of DNA topology. Its catalytic function is to remove positive or negative superhelical tension by binding to duplex DNA, creating a reversible single-strand break, and finally religating the broken strand. Proper maintenance of DNA topological homeostasis, in turn, is critically important in the regulation of replication, transcription, DNA repair, and other processes of DNA metabolism. One of the cellular processes regulated by the DNA topology and thus by Topoisomerase I is the formation of non-canonical DNA structures. Non-canonical or non-B DNA structures, including the four-stranded G-quadruplex or G4 DNA, are potentially pathological in that they interfere with replication or transcription, forming hotspots of genome instability. In this review, we first describe the role of Topoisomerase I in reducing the formation of non-canonical nucleic acid structures in the genome. We further discuss the interesting recent discovery that Top1 and Top1 mutants bind to G4 DNA structures in vivo and in vitro and speculate on the possible consequences of these interactions.

Response of Sulfolobus solfataricus Dpo4 polymerase in vitro to a DNA G-quadruplex.

Repetitive DNA sequences support the formation of structures that can interrupt replication and repair, leading to breaks and mutagenesis. One particularly stable structure is G-quadruplex (G4) DNA, which is four-stranded and formed from tandemly repetitive guanine bases. When folded within a template, G4 interferes with DNA synthesis. Similar to non-duplex structures, DNA base lesions can also halt an advancing replication fork, but the Y-family polymerases solve this problem by bypassing the damage. In order to better understand how guanine-rich DNA is replicated, we have investigated the activity of the model Y-family polymerase, Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4), on guanine-rich templates in vitro. We find that Dpo4 progression on templates containing either a single GC-rich hairpin or a G4 DNA structure is greatly reduced and synthesis stalls at the structure. Human polymerase eta (hPol eta) showed the same pattern of stalling at G4; however, and in contrast to Klenow, hPol eta and Dpo4 partially synthesise into the guanine repeat. Substitution of the nucleotide selectivity residue in Dpo4 with alanine permitted ribonucleotide incorporation on unstructured templates, but this further reduced the ability of Dpo4 to synthesise across from the guanine repeats. The advancement of Dpo4 on G4 templates was highest when the reaction was supplied with only deoxycytidine triphosphate, suggesting that high-fidelity synthesis is favoured over misincorporation. Our results are consistent with a model where the Y-family polymerases pause upon encountering G4 structures but have an ability to negotiate some synthesis through tetrad-associated guanines. This suggests that the Y-family polymerases reduce mutagenesis by catalysing the accurate replication of repetitive DNA sequences, but most likely in concert with additional replication and structure resolution activities.

Sites of instability in the human TCF3 (E2A) gene adopt G-quadruplex DNA structures in vitro.

The formation of highly stable four-stranded DNA, called G-quadruplex (G4), promotes site-specific genome instability. G4 DNA structures fold from repetitive guanine sequences, and increasing experimental evidence connects G4 sequence motifs with specific gene rearrangements. The human transcription factor 3 (TCF3) gene (also termed E2A) is subject to genetic instability associated with severe disease, most notably a common translocation event t(1;19) associated with acute lymphoblastic leukemia. The sites of instability in TCF3 are not randomly distributed, but focused to certain sequences. We asked if G4 DNA formation could explain why TCF3 is prone to recombination and mutagenesis. Here we demonstrate that sequences surrounding the major t(1;19) break site and a region associated with copy number variations both contain G4 sequence motifs. The motifs identified readily adopt G4 DNA structures that are stable enough to interfere with DNA synthesis in physiological salt conditions in vitro. When introduced into the yeast genome, TCF3 G4 motifs promoted gross chromosomal rearrangements in a transcription-dependent manner. Our results provide a molecular rationale for the site-specific instability of human TCF3, suggesting that G4 DNA structures contribute to oncogenic DNA breaks and recombination.