Impact of G-Quadruplexes on the Regulation of Genome Integrity, DNA Damage and Repair

DNA G-quadruplexes (G4s) are known to be an integral part of the complex regulatory systems in both normal and pathological cells. At the same time, the ability of G4s to impede DNA replication plays a critical role in genome integrity. This review summarizes the results of recent studies of G4-mediated genomic and epigenomic instability, together with associated DNA damage and repair processes. Although the underlying mechanisms remain to be elucidated, it is known that, among the proteins that recognize G4 structures, many are linked to DNA repair. We analyzed the possible role of G4s in promoting double-strand DNA breaks, one of the most deleterious DNA lesions, and their repair via error-prone mechanisms. The patterns of G4 damage, with a focus on the introduction of oxidative guanine lesions, as well as their removal from G4 structures by canonical repair pathways, were also discussed together with the effects of G4s on the repair machinery. According to recent findings, there must be a delicate balance between G4-induced genome instability and G4-promoted repair processes. A broad overview of the factors that modulate the stability of G4 structures in vitro and in vivo is also provided here

New DNA Plasmid Model for Studying DNA Mismatch Repair Response to the G4 Structure

G-quadruplexes (G4s), the most widely studied alternative DNA structures, are implicated in the regulation of the key cellular processes. In recent years, their involvement in DNA repair machinery has become the subject of intense research. Here, we evaluated the effect of G4 on the prokaryotic DNA mismatch repair (MMR) pathway from two bacterial sources with different mismatch repair mechanisms. The G4 folding, which competes with the maintenance of double-stranded DNA, is known to be controlled by numerous opposing factors. To overcome the kinetic barrier of G4 formation, we stabilized a parallel G4 formed by the d(GGGT)4 sequence in a DNA plasmid lacking a fragment complementary to the G4 motif. Unlike commonly used isolated G4 structures, our plasmid with an embedded stable G4 structure contained elements, such as a MutH cleavage site, required to initiate the repair process. G4 formation in the designed construct was confirmed by Taq polymerase stop assay and dimethyl sulfate probing. The G4-carrying plasmid, together with control ones (lacking a looped area or containing unstructured d(GT)8 insert instead of the G4 motif), were used as new type models to answer the question of whether G4 formation interferes with DNA cleavage as a basic function of MMR

Synthetic Hexanucleotides as a Tool to Overcome Excessive Neutrophil Activation Caused by CpG-Containing Oligonucleotides

Mimicking bacterial DNA, synthetic CpG-containing oligodeoxyribonucleotides (CpG-ODNs) have a powerful immunomodulatory potential. Their practical application is mainly associated with the production of vaccines, where they are used as adjuvants, as well as in local antimicrobial therapy. CpG-ODNs act on a wide variety of immune cells, including neutrophilic granulocytes. On the one hand, the stimulatory effect provides both the direct implementation of their antimicrobial and fungicidal mechanisms, and an avalanche-like strengthening of the immune signal due to interaction with other participants in the immune process. On the other hand, hyperactivation of neutrophilic granulocytes can have negative consequences. In particular, the formation of unreasonably high amounts of reactive oxygen species leads to tissue damages and, as a consequence, a spontaneous aggravation and prolongation of the inflammatory process. Under physiological conditions, a large number of DNA fragments are present in inflammation foci: both of microbial and self-tissue origin. We investigated effects of several short modified hexanucleotides on the main indicators of neutrophil activation, as well as their influence on the immunomodulatory activity of known synthetic CpG-ODNs. The results obtained show that short oligonucleotides partially inhibit the prooxidant effect of synthetic CpG-ODNs without significantly affecting the ability of the latter to overcome bacteria-induced pro-survival effects on neutrophilic granulocytes

Impact of G-Quadruplex Structures on Methylation of Model Substrates by DNA Methyltransferase Dnmt3a

In mammals, de novo methylation of cytosines in DNA CpG sites is performed by DNA methyltransferase Dnmt3a. Changes in the methylation status of CpG islands are critical for gene regulation and for the progression of some cancers. Recently, the potential involvement of DNA G-quadruplexes (G4s) in methylation control has been found. Here, we provide evidence for a link between G4 formation and the function of murine DNA methyltransferase Dnmt3a and its individual domains. As DNA models, we used (i) an isolated G4 formed by oligonucleotide capable of folding into parallel quadruplex and (ii) the same G4 inserted into a double-stranded DNA bearing several CpG sites. Using electrophoretic mobility shift and fluorescence polarization assays, we showed that the Dnmt3a catalytic domain (Dnmt3a-CD), in contrast to regulatory PWWP domain, effectively binds the G4 structure formed in both DNA models. The G4-forming oligonucleotide displaced the DNA substrate from its complex with Dnmt3a-CD, resulting in a dramatic suppression of the enzyme activity. In addition, a direct impact of G4 inserted into the DNA duplex on the methylation of a specific CpG site was revealed. Possible mechanisms of G4-mediated epigenetic regulation may include Dnmt3a sequestration at G4 and/or disruption of Dnmt3a oligomerization on the DNA surface

Magic Peptide: Unique Properties of the LRR11 Peptide in the Activation of Leukotriene Synthesis in Human Neutrophils

Neutrophil-mediated innate host defense mechanisms include pathogen elimination through bacterial phagocytosis, which activates the 5-lipoxygenase (5-LOX) product synthesis. Here, we studied the effect of synthetic oligodeoxyribonucleotides (ODNs), which mimic the receptor-recognized sites of bacterial (CpG-ODNs) and genomic (G-rich ODNs) DNAs released from the inflammatory area, on the neutrophil functions after cell stimulation with Salmonella typhimurium. A possible mechanism for ODN recognition by Toll-like receptor 9 (TLR9) and RAGE receptor has been proposed. We found for the first time that the combination of the magic peptide LRR11 from the leucine-rich repeat (LRR) of TLR9 with the CpG-ODNs modulates the uptake and signaling from ODNs, in particular, dramatically stimulates 5-LOX pathway. Using thickness shear mode acoustic method, we confirmed the specific binding of CpG-ODNs, but not G-rich ODN, to LRR11. The RAGE receptor has been shown to play an important role in promoting ODN uptake. Thus, FPS-ZM1, a high-affinity RAGE inhibitor, suppresses the synthesis of 5-LOX products and reduces the uptake of ODNs by neutrophils; the inhibitor effect being abolished by the addition of LRR11. The results obtained revealed that the studied peptide-ODN complexes possess high biological activity and can be promising for the development of effective vaccine adjuvants and antimicrobial therapeutics

G-Quadruplex Formed by the Promoter Region of the hTERT Gene: Structure-Driven Effects on DNA Mismatch Repair Functions

G-quadruplexes (G4s) are a unique class of noncanonical DNAs that play a key role in cellular processes and neoplastic transformation. Herein, we focused on the promoter region of human TERT oncogene, whose product is responsible for the immortality of cancer cells. It has been shown by chemical probing and spectroscopic methods that synthetic 96-nt DNAs modeling the wild-type G-rich strand of the hTERT promoter and its variants with G>A point substitutions corresponding to somatic driver mutations fold into three stacked parallel G4s with sites of local G4 destabilization caused by G>A substitutions in the G4 motif. These models were used to elucidate how the hTERT multiG4 affects the binding affinity and functional responses of two key proteins, MutS and MutL, involved in the initial stage of DNA mismatch repair (MMR) in Escherichiacoli and Neisseriagonorrhoeae with different MMR mechanisms. We have shown for the first time that (i) point substitutions do not affect the effective binding of these proteins to the hTERT G4 structure, and (ii) the endonuclease activity of MutL from N. gonorrhoeae is significantly suppressed by the stable G4 scaffold. It is likely that some of the genomic instability associated with G4 may be related to the blockage of human intrinsic methyl-independent MMR attempting to operate near G4 structures