6n2t | pdb_00006n2t
Ternary complex crystal structure of DNA polymerase Beta with 5-hydroxymethyl-dC (5-hmC) at the templating position
Structural highlights
Function[DPOLB_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 PubMedDNA methylation is an epigenetic mark that regulates gene expression in mammals. One method of methylation removal is through TET-catalyzed oxidation and the base excision repair pathway. The iterative oxidation of 5-methylcytosine (5mC) catalyzed by TET enzymes produces three oxidized forms of cytosine: 5-hydroxmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC). The effect these modifications have on the efficiency and fidelity of the base excision repair pathway during the repair of opposing base damage, and in particular DNA polymerization, remains to be elucidated. Using kinetic assays, we show that the catalytic efficiency for the incorporation of dGTP catalyzed by human DNA Polymerase beta is not affected when 5mC, 5hmC, 5fC are in the DNA template. In contrast, the catalytic efficiency of dGTP insertion decreases ~20-fold when 5caC is in the templating position, as compared to unmodified cytosine. However, DNA polymerase fidelity is unaltered when these modifications are in the templating position. Structural analysis reveals that the methyl, hydroxymethyl, and formyl modifications are easily accommodated within the polymerase active site. However, to accommodate the carboxy modification, the phosphate backbone on the templating nucleotide shifts ~2.5 A to avoid a potential steric/repulsive clash. This altered conformation is stabilized by lysine 280 which makes a direct interaction with the carboxy modification and the phosphate backbone of the templating strand. This work provides the molecular basis for the accommodation of epigenetic base modifications in a polymerase active site and suggests that these modifications are not mutagenically copied during BER. Molecular basis for the faithful replication of 5-methylcytosine and its oxidized forms by DNA Polymerase ss.,Howard MJ, Foley KG, Shock DD, Batra VK, Wilson SH J Biol Chem. 2019 Mar 18. pii: RA118.006809. doi: 10.1074/jbc.RA118.006809. PMID:30885943[5] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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