Synthesis, base pairing properties and trans-lesion synthesis by reverse transcriptases of oligoribonucleotides containing the oxidatively damaged base 5-hydroxycytidine

Küpfer, Pascal A.; Leumann, Christian J. (2011). Synthesis, base pairing properties and trans-lesion synthesis by reverse transcriptases of oligoribonucleotides containing the oxidatively damaged base 5-hydroxycytidine. Nucleic acids research, 39(21), pp. 9422-9432. London: Oxford University Press 10.1093/nar/gkr673

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The synthesis of a caged RNA phosphoramidite building block containing the oxidatively damaged base 5-hydroxycytidine (5-HOrC) has been accomplished. To determine the effect of this highly mutagenic lesion on complementary base recognition and coding properties, this building block was incorporated into a 12-mer oligoribonucleotide for Tm and CD measurements and a 31-mer template strand for primer extension experiments with HIV-, AMV- and MMLV-reverse transcriptase (RT). In UV-melting experiments, we find an unusual biphasic transition with two distinct Tm's when 5-HOrC is paired against a DNA or RNA complement with the base guanine in opposing position. The higher Tm closely matches that of a C-G base pair while the lower is close to that of a C-A mismatch. In single nucleotide extension reactions, we find substantial misincorporation of dAMP and to a lesser extent dTMP, with dAMP almost equaling that of the parent dGMP in the case of HIV-RT. A working hypothesis for the biphasic melting transition does not invoke tautomeric variability of 5-HOrC but rather local structural perturbations of the base pair at low temperature induced by interactions of the 5-HO group with the phosphate backbone. The properties of this RNA damage is discussed in the context of its putative biological function.

Item Type:

Journal Article (Original Article)

Division/Institute:

08 Faculty of Science > Departement of Chemistry and Biochemistry

UniBE Contributor:

Küpfer, Pascal and Leumann, Christian

Subjects:

500 Science > 570 Life sciences; biology
500 Science > 540 Chemistry

ISSN:

0305-1048

Publisher:

Oxford University Press

Language:

English

Submitter:

Christian Leumann

Date Deposited:

04 Oct 2013 14:26

Last Modified:

23 Oct 2019 09:21

Publisher DOI:

10.1093/nar/gkr673

Web of Science ID:

000297375700039

BORIS DOI:

10.7892/boris.9414

URI:

https://boris.unibe.ch/id/eprint/9414 (FactScience: 215146)

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