6UOL

Y271G DNA polymerase beta substrate complex with a templating cytosine and incoming rGTP

Summary for 6UOL

• Entry DOI: 10.2210/pdb6uol/pdb
• Descriptor: DNA (5'-D(*CP*CP*GP*AP*CP*CP*GP*CP*GP*CP*AP*TP*CP*AP*GP*C)-3'), DNA (5'-D(*GP*CP*TP*GP*AP*TP*GP*CP*GP*C)-3'), DNA (5'-D(P*GP*TP*CP*GP*G)-3'), ... (10 entities in total)
• Functional Keywords: dna polymerase, ribonucleotide, oxidized nucleotide, dna damage, dna binding protein, transferase-dna complex, transferase/dna
• Biological source: Homo sapiens (Human); More
• Total number of polymer chains: 4
• Total formula weight: 48510.09

Authors

Smith, M.R.,Freudenthal, B.D. (deposition date: 2019-10-15, release date: 2020-01-08, Last modification date: 2023-10-11)

Primary citation

Smith, M.R.,Alnajjar, K.S.,Hoitsma, N.M.,Sweasy, J.B.,Freudenthal, B.D.
Molecular and structural characterization of oxidized ribonucleotide insertion into DNA by human DNA polymerase beta.
J.Biol.Chem., 295:1613-1622, 2020

PubMed 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 ( 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 conformation opposite a templating cytosine and a 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.

PubMed: 31892517
DOI: 10.1074/jbc.RA119.011569

Experimental method

X-RAY DIFFRACTION (1.936 Å)