Cilli Piera, Minoprio Anna, Bossa Cecilia, Bignami Margherita, Mazzei Filomena
From the Department of Environment and Primary Prevention, Istituto Superiore di Sanità, 00161 Roma and the Department of Science, University Roma Tre, 00154 Roma, Italy.
From the Department of Environment and Primary Prevention, Istituto Superiore di Sanità, 00161 Roma and.
J Biol Chem. 2015 Oct 23;290(43):26259-69. doi: 10.1074/jbc.M115.679209. Epub 2015 Sep 3.
The cellular pool of ribonucleotide triphosphates (rNTPs) is higher than that of deoxyribonucleotide triphosphates. To ensure genome stability, DNA polymerases must discriminate against rNTPs and incorporated ribonucleotides must be removed by ribonucleotide excision repair (RER). We investigated DNA polymerase β (POL β) capacity to incorporate ribonucleotides into trinucleotide repeated DNA sequences and the efficiency of base excision repair (BER) and RER enzymes (OGG1, MUTYH, and RNase H2) when presented with an incorrect sugar and an oxidized base. POL β incorporated rAMP and rCMP opposite 7,8-dihydro-8-oxoguanine (8-oxodG) and extended both mispairs. In addition, POL β was able to insert and elongate an oxidized rGMP when paired with dA. We show that RNase H2 always preserves the capacity to remove a single ribonucleotide when paired to an oxidized base or to incise an oxidized ribonucleotide in a DNA duplex. In contrast, BER activity is affected by the presence of a ribonucleotide opposite an 8-oxodG. In particular, MUTYH activity on 8-oxodG:rA mispairs is fully inhibited, although its binding capacity is retained. This results in the reduction of RNase H2 incision capability of this substrate. Thus complex mispairs formed by an oxidized base and a ribonucleotide can compromise BER and RER in repeated sequences.
核糖核苷酸三磷酸（rNTPs）的细胞池比脱氧核糖核苷酸三磷酸的细胞池更大。为确保基因组稳定性，DNA聚合酶必须区分rNTPs，并且掺入的核糖核苷酸必须通过核糖核苷酸切除修复（RER）去除。我们研究了DNA聚合酶β（POL β）将核糖核苷酸掺入三核苷酸重复DNA序列的能力，以及当存在错误糖和氧化碱基时碱基切除修复（BER）和RER酶（OGG1、MUTYH和核糖核酸酶H2）的效率。POL β在7,8-二氢-8-氧代鸟嘌呤（8-氧代dG）对面掺入rAMP和rCMP，并延伸了这两种错配。此外，当与dA配对时，POL β能够插入并延长氧化的rGMP。我们发现，核糖核酸酶H2在与氧化碱基配对时始终保持去除单个核糖核苷酸的能力，或在DNA双链体中切割氧化的核糖核苷酸。相比之下，BER活性受8-氧代dG对面核糖核苷酸存在的影响。特别是，MUTYH对8-氧代dG:rA错配的活性被完全抑制，尽管其结合能力得以保留。这导致该底物的核糖核酸酶H2切割能力降低。因此，由氧化碱基和核糖核苷酸形成的复杂错配会损害重复序列中的BER和RER。
