6uom
From Proteopedia
Y271G DNA polymerase beta ternary complex with templating adenine and incoming r8-oxo-GTP
Structural highlights
FunctionDPOLB_HUMAN Repair polymerase that plays a key role in base-excision repair. Has 5'-deoxyribose-5-phosphate lyase (dRP lyase) activity that removes the 5' sugar phosphate and also acts as a DNA polymerase that adds one nucleotide to the 3' end of the arising single-nucleotide gap. Conducts 'gap-filling' DNA synthesis in a stepwise distributive fashion rather than in a processive fashion as for other DNA polymerases.[1] [2] [3] [4] Publication Abstract from PubMedDuring 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 beta (pol beta) and characterized r8-oxo-GTP insertion with DNA substrates containing either a templating cytosine (nonmutagenic) or adenine (mutagenic). Our results show that pol beta 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 beta with r8-oxo-GTP, we demonstrate impaired pol beta 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. Molecular and structural characterization of oxidized ribonucleotide insertion into DNA by human DNA polymerase beta.,Smith MR, Alnajjar KS, Hoitsma NM, Sweasy JB, Freudenthal BD J Biol Chem. 2020 Feb 7;295(6):1613-1622. doi: 10.1074/jbc.RA119.011569. Epub, 2019 Dec 31. PMID:31892517[5] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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