Summary information and primary citation

6uol; DSSR-derived features in text and JSON formats
X-ray (1.936 Å)
Y271g DNA polymerase beta substrate complex with a templating cytosine and incoming rgtp
Smith MR, Alnajjar KS, Hoitsma NM, Sweasy JB, Freudenthal BD (2020): "Molecular and structural characterization of oxidized ribonucleotide insertion into DNA by human DNA polymerase beta." J.Biol.Chem., 295, 1613-1622. doi: 10.1074/jbc.RA119.011569.
During oxidative stress, inflammation, or environmental exposures, 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 (non-mutagenic) 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 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.

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