Impact of 1,N6-ethenoadenosine, a damaged ribonucleotide in DNA, on translesion synthesis and repair
Impact of 1,N6-ethenoadenosine, a damaged ribonucleotide in DNA, on translesion synthesis and repair
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DOI：
10.1074/jbc.ra120.012829
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发表时间：
2020-05-01
影响因子：
4.8
通讯作者：
Guengerich, F. Peter
中科院分区：
文献类型：
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作者：
Ghodke, Pratibha P.;Guengerich, F. Peter
核糖核苷酸掺入DNA会严重破坏基因组的完整性。然而，核糖核苷酸如何引发DNA损伤的了解甚少。在DNA中，它们可以促进复制应激和基因组不稳定性，并与几种疾病有关。我们在这里报告了核糖核苷酸rATP及其天然存在的受损类似物1，N-6-乙烯基腺苷（1，N-6-？rA）对人DNA聚合酶介导的跨损伤合成（TLS）的影响？(hpol?),在RNase H2？中间切口质谱分析表明，1，N-6-？DNA中的rA在TLS期间产生广泛的移码，这可能导致基因组不稳定。此外，稳态动力学分析的TLS过程表明，脱氧嘌呤（即dATP和dGTP）插入主要是相反的1，N-6-？RA.我们还表明，hpol？作为一种逆转录酶的存在下，受损的核糖核苷酸1，N-6-？但具有差的RNA引物延伸活性。逆转录和RNA引物延伸的稳态动力学分析表明，hpol？有利于添加dATP和dGTP相反1，N-6-？RA.我们还发现，RNase H2识别1，N-6-？rA，但在该损伤对面具有有限的切割活性，这可能导致这种有害的DNA加合物的持续存在。我们的结论是，受损和未修复的核糖核苷酸1，N-6-？DNA中的rA具有致突变的潜力，也可以改变mRNA转录物中的阅读框架，因为1，N-6-？rA被RNase H2不完全切割。
Incorporation of ribonucleotides into DNA can severely diminish genome integrity. However, how ribonucleotides instigate DNA damage is poorly understood. In DNA, they can promote replication stress and genomic instability and have been implicated in several diseases. We report here the impact of the ribonucleotide rATP and of its naturally occurring damaged analog 1,N-6-ethenoadenosine (1,N-6-?rA) on translesion synthesis (TLS), mediated by human DNA polymerase ? (hpol ?), and on RNase H2?mediated incision. Mass spectral analysis revealed that 1,N-6-?rA in DNA generates extensive frameshifts during TLS, which can lead to genomic instability. Moreover, steady-state kinetic analysis of the TLS process indicated that deoxypurines (i.e. dATP and dGTP) are inserted predominantly opposite 1,N-6-?rA. We also show that hpol ? acts as a reverse transcriptase in the presence of damaged ribonucleotide 1,N-6-?rA but has poor RNA primer extension activities. Steady-state kinetic analysis of reverse transcription and RNA primer extension showed that hpol ? favors the addition of dATP and dGTP opposite 1,N-6-?rA. We also found that RNase H2 recognizes 1,N-6-?rA but has limited incision activity across from this lesion, which can lead to the persistence of this detrimental DNA adduct. We conclude that the damaged and unrepaired ribonucleotide 1,N-6-?rA in DNA exhibits mutagenic potential and can also alter the reading frame in an mRNA transcript because 1,N-6-?rA is incompletely incised by RNase H2.