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Site-specific stabilization of DNA by a tethered major groove amine, 7-aminomethyl-7-deaza-2'-deoxyguanosine.

ABSTRACT: A cationic 7-aminomethyl-7-deaza-2'-deoxyguanosine (7amG) was incorporated site-specifically into the self-complementary duplex d(G¹A²G³A?X?C?G?C?T?C¹?T¹¹C¹²)? (X = 7amG). This construct placed two positively charged amines adjacent to the major groove edges of two symmetry-related guanines, providing a model for probing how cation binding in the major groove modulates the structure and stability of DNA. Molecular dynamics calculations restrained by nuclear magnetic resonance (NMR) data revealed that the tethered cationic amines were in plane with the modified base pairs. The tethered amines did not form salt bridges to the phosphodiester backbone. There was also no indication of the amines being capable of hydrogen bonding to flanking DNA bases. NMR spectroscopy as a function of temperature revealed that the X? imino resonance remained sharp at 55 °C. Additionally, two 5'-neighboring base pairs, A?:T? and G³:C¹?, were stabilized with respect to the exchange of their imino protons with solvent. The equilibrium constant for base pair opening at the A?:T? base pair determined by magnetization transfer from water in the absence and presence of added ammonia base catalyst decreased for the modified duplex compared to that of the A?:T? base pair in the unmodified duplex, which confirmed that the overall fraction of the A?:T? base pair in the open state of the modified duplex decreased. This was also observed for the G³:C¹? base pair, where ?K(op) for the G³:C¹? base pair in the modified duplex was 3.0 × 10? versus 4.1 × 10? for the same base pair in the unmodified duplex. In contrast, equilibrium constants for base pair opening at the X?:C? and C?:G? base pairs did not change at 15 °C. These results argue against the notion that electrostatic interactions with DNA are entirely entropic and suggest that major groove cations can stabilize DNA via enthalpic contributions to the free energy of duplex formation.

SUBMITTER: Szulik MW

PROVIDER: S-EPMC3812902 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Site-specific stabilization of DNA by a tethered major groove amine, 7-aminomethyl-7-deaza-2'-deoxyguanosine.

Szulik Marta W MW Voehler Markus W MW Ganguly Manjori M Gold Barry B Stone Michael P MP

Biochemistry 20131016 43

A cationic 7-aminomethyl-7-deaza-2'-deoxyguanosine (7amG) was incorporated site-specifically into the self-complementary duplex d(G¹A²G³A⁴X⁵C⁶G⁷C⁸T⁹C¹⁰T¹¹C¹²)₂ (X = 7amG). This construct placed two positively charged amines adjacent to the major groove edges of two symmetry-related guanines, providing a model for probing how cation binding in the major groove modulates the structure and stability of DNA. Molecular dynamics calculations restrained by nuclear magnetic resonance (NMR) data revealed ...[more]

PMID: 24131376

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Project description:Several reports have demonstrated that Campylobacter bacteriophage DNA is refractory to manipulation, suggesting that these phages encode modified DNA. The characterized Campylobacter jejuni phages fall into two phylogenetic groups within the Myoviridae: the genera Firehammervirus and Fletchervirus Analysis of genomic nucleosides from several of these phages by high-pressure liquid chromatography-mass spectrometry confirmed that 100% of the 2'-deoxyguanosine (dG) residues are replaced by modified bases. Fletcherviruses replace dG with 2'-deoxyinosine, while the firehammerviruses replace dG with 2'-deoxy-7-amido-7-deazaguanosine (dADG), noncanonical nucleotides previously described, but a 100% base substitution has never been observed to have been made in a virus. We analyzed the genome sequences of all available phages representing both groups to elucidate the biosynthetic pathway of these noncanonical bases. Putative ADG biosynthetic genes are encoded by the Firehammervirus phages and functionally complement mutants in the Escherichia coli queuosine pathway, of which ADG is an intermediate. To investigate the mechanism of DNA modification, we isolated nucleotide pools and identified dITP after phage infection, suggesting that this modification is made before nucleotides are incorporated into the phage genome. However, we were unable to observe any form of dADG phosphate, implying a novel mechanism of ADG incorporation into an existing DNA strand. Our results imply that Fletchervirus and Firehammervirus phages have evolved distinct mechanisms to express dG-free DNA.IMPORTANCE Bacteriophages are in a constant evolutionary struggle to overcome their microbial hosts' defenses and must adapt in unconventional ways to remain viable as infectious agents. One mode of adaptation is modifying the viral genome to contain noncanonical nucleotides. Genome modification in phages is becoming more commonly reported as analytical techniques improve, but guanosine modifications have been underreported. To date, two genomic guanosine modifications have been observed in phage genomes, and both are low in genomic abundance. The significance of our research is in the identification of two novel DNA modification systems in Campylobacter-infecting phages, which replace all guanosine bases in the genome in a genus-specific manner.

Dataset Information

Site-specific stabilization of DNA by a tethered major groove amine, 7-aminomethyl-7-deaza-2'-deoxyguanosine.

Publications

Site-specific stabilization of DNA by a tethered major groove amine, 7-aminomethyl-7-deaza-2'-deoxyguanosine.

Similar Datasets

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