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Molecular and structural characterization of oxidized ribonucleotide insertion into DNA by human DNA polymerase β.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2020 Feb 07; Vol. 295 (6), pp. 1613-1622. Date of Electronic Publication: 2019 Dec 31. - Publication Year :
- 2020
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Abstract
- During oxidative stress, inflammation, or environmental exposure, ribo- and deoxyribonucleotides are oxidatively modified. 8-Oxo-7,8-dihydro-2'-guanosine (8-oxo-G) is a common oxidized nucleobase whose deoxyribonucleotide form, 8-oxo-dGTP, has been widely studied and demonstrated to be a mutagenic substrate for DNA polymerases. Guanine ribonucleotides are analogously oxidized to r8-oxo-GTP, which can constitute up to 5% of the rGTP pool. Because ribonucleotides are commonly misinserted into DNA, and 8-oxo-G causes replication errors, we were motivated to investigate how the oxidized ribonucleotide is utilized by DNA polymerases. To do this, here we employed human DNA polymerase β (pol β) and characterized r8-oxo-GTP insertion with DNA substrates containing either a templating cytosine (nonmutagenic) or adenine (mutagenic). Our results show that pol β has a diminished catalytic efficiency for r8-oxo-GTP compared with canonical deoxyribonucleotides but that r8-oxo-GTP is inserted mutagenically at a rate similar to those of other common DNA replication errors ( i.e. ribonucleotide and mismatch insertions). Using FRET assays to monitor conformational changes of pol β with r8-oxo-GTP, we demonstrate impaired pol β closure that correlates with a reduced insertion efficiency. X-ray crystallographic analyses revealed that, similar to 8-oxo-dGTP, r8-oxo-GTP adopts an anti conformation opposite a templating cytosine and a syn conformation opposite adenine. However, unlike 8-oxo-dGTP, r8-oxo-GTP did not form a planar base pair with either templating base. These results suggest that r8-oxo-GTP is a potential mutagenic substrate for DNA polymerases and provide structural insights into how r8-oxo-GTP is processed by DNA polymerases.<br /> (© 2020 Smith et al.)
Details
- Language :
- English
- ISSN :
- 1083-351X
- Volume :
- 295
- Issue :
- 6
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 31892517
- Full Text :
- https://doi.org/10.1074/jbc.RA119.011569