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Transcriptional Perturbations of 2,6-Diaminopurine and 2-Aminopurine.

ABSTRACT: 2,6-Diaminopurine (Z) is a naturally occurring adenine (A) analog that bacteriophages employ in place of A in their genetic alphabet. Recent discoveries of biogenesis pathways of Z in bacteriophages have stimulated substantial research interest in this DNA modification. Here, we systematically examined the effects of Z on the efficiency and fidelity of DNA transcription. Our results showed that Z exhibited no mutagenic yet substantial inhibitory effects on transcription mediated by purified T7 RNA polymerase and by human RNA polymerase II in HeLa nuclear extracts and in human cells. A structurally related adenine analog, 2-aminopurine (2AP), strongly blocked T7 RNA polymerase but did not impede human RNA polymerase II in vitro or in human cells, where no mutant transcript could be detected. The lack of mutagenic consequence and the presence of a strong blockage effect of Z on transcription suggest a role of Z in transcriptional regulation. Z is also subjected to removal by transcription-coupled nucleotide-excision repair (TC-NER), but not global-genome NER in human cells. Our findings provide new insight into the effects of Z on transcription and its potential biological functions.

SUBMITTER: Tan Y

PROVIDER: S-EPMC9354233 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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Transcriptional Perturbations of 2,6-Diaminopurine and 2-Aminopurine.

Tan Ying Y You Changjun C Park Jiyeong J Kim Hyun Suk HS Guo Su S Schärer Orlando D OD Wang Yinsheng Y

ACS chemical biology 20220614 7

2,6-Diaminopurine (Z) is a naturally occurring adenine (A) analog that bacteriophages employ in place of A in their genetic alphabet. Recent discoveries of biogenesis pathways of Z in bacteriophages have stimulated substantial research interest in this DNA modification. Here, we systematically examined the effects of Z on the efficiency and fidelity of DNA transcription. Our results showed that Z exhibited no mutagenic yet substantial inhibitory effects on transcription mediated by purified T7 R ...[more]

PMID: 35700389

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Project description:2,6-diaminopurine (DAP) is a nucleobase analog of adenine. When incorporated into double-stranded DNA (dsDNA), it forms three hydrogen bonds with thymine. Rare in nature, DAP substitution alters the physical characteristics of a DNA molecule without sacrificing sequence specificity. Here, we show that in addition to stabilizing double-strand hybridization, DAP substitution also changes the mechanical and conformational properties of dsDNA. Thermal melting experiments reveal that DAP substitution raises melting temperatures without diminishing sequence-dependent effects. Using a combination of atomic force microscopy (AFM), magnetic tweezer (MT) nanomechanical assays, and circular dichroism spectroscopy, we demonstrate that DAP substitution increases the flexural rigidity of dsDNA yet also facilitates conformational shifts, which manifest as changes in molecule length. DAP substitution increases both the static and dynamic persistence length of DNA (measured by AFM and MT, respectively). In the static case (AFM), in which tension is not applied to the molecule, the contour length of DAP-DNA appears shorter than wild-type (WT)-DNA; under tension (MT), they have similar dynamic contour lengths. At tensions above 60 pN, WT-DNA undergoes characteristic overstretching because of strand separation (tension-induced melting) and spontaneous adoption of a conformation termed S-DNA. Cyclic overstretching and relaxation of WT-DNA at near-zero loading rates typically yields hysteresis, indicative of tension-induced melting; conversely, cyclic stretching of DAP-DNA showed little or no hysteresis, consistent with the adoption of the S-form, similar to what has been reported for GC-rich sequences. However, DAP-DNA overstretching is distinct from GC-rich overstretching in that it happens at a significantly lower tension. In physiological salt conditions, evenly mixed AT/GC DNA typically overstretches around 60 pN. GC-rich sequences overstretch at similar if not slightly higher tensions. Here, we show that DAP-DNA overstretches at 52 pN. In summary, DAP substitution decreases the overall stability of the B-form double helix, biasing toward non-B-form DNA helix conformations at zero tension and facilitating the B-to-S transition at high tension.

Dataset Information

Transcriptional Perturbations of 2,6-Diaminopurine and 2-Aminopurine.

Publications

Transcriptional Perturbations of 2,6-Diaminopurine and 2-Aminopurine.

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