Hoopes James I, Hughes Amber L, Hobson Lauren A, Cortez Luis M, Brown Alexander J, Roberts Steven A
School of Molecular Biosciences, College of Veterinary Medicine, Washington State University, Pullman, WA 99164, USA.
Nucleic Acids Res. 2017 May 19;45(9):5243-5254. doi: 10.1093/nar/gkx169.
APOBEC cytidine deaminases mutate cancer genomes by converting cytidines into uridines within ssDNA during replication. Although uracil DNA glycosylases limit APOBEC-induced mutation, it is unknown if subsequent base excision repair (BER) steps function on replication-associated ssDNA. Hence, we measured APOBEC3B-induced CAN1 mutation frequencies in yeast deficient in BER endonucleases or DNA damage tolerance proteins. Strains lacking Apn1, Apn2, Ntg1, Ntg2 or Rev3 displayed wild-type frequencies of APOBEC3B-induced canavanine resistance (CanR). However, strains without error-free lesion bypass proteins Ubc13, Mms2 and Mph1 displayed respective 4.9-, 2.8- and 7.8-fold higher frequency of APOBEC3B-induced CanR. These results indicate that mutations resulting from APOBEC activity are avoided by deoxyuridine conversion to abasic sites ahead of nascent lagging strand DNA synthesis and subsequent bypass by error-free template switching. We found this mechanism also functions during telomere re-synthesis, but with a diminished requirement for Ubc13. Interestingly, reduction of G to C substitutions in Ubc13-deficient strains uncovered a previously unknown role of Ubc13 in controlling the activity of the translesion synthesis polymerase, Rev1. Our results highlight a novel mechanism for error-free bypass of deoxyuridines generated within ssDNA and suggest that the APOBEC mutation signature observed in cancer genomes may under-represent the genomic damage these enzymes induce.
载脂蛋白B mRNA编辑酶催化多肽样胞嘧啶脱氨酶（APOBEC）胞嘧啶脱氨酶在复制过程中通过将单链DNA（ssDNA）中的胞嘧啶转化为尿嘧啶来使癌症基因组发生突变。尽管尿嘧啶DNA糖基化酶限制了APOBEC诱导的突变，但尚不清楚随后的碱基切除修复（BER）步骤是否作用于与复制相关的ssDNA。因此，我们在缺乏BER核酸内切酶或DNA损伤耐受蛋白的酵母中测量了APOBEC3B诱导的CAN1突变频率。缺乏Apn1、Apn2、Ntg1、Ntg2或Rev3的菌株显示出APOBEC3B诱导的刀豆氨酸抗性（CanR）的野生型频率。然而，没有无差错损伤旁路蛋白Ubc13、Mms2和Mph1的菌株显示出APOBEC3B诱导的CanR频率分别高出4.9倍、2.8倍和7.8倍。这些结果表明，通过在新生滞后链DNA合成之前将脱氧尿苷转化为无碱基位点并随后通过无差错模板转换进行旁路，可以避免APOBEC活性导致的突变。我们发现这种机制在端粒重新合成过程中也起作用，但对Ubc13的需求减少。有趣的是，Ubc13缺陷菌株中G到C替换的减少揭示了Ubc13在控制跨损伤合成聚合酶Rev1活性方面以前未知的作用。我们的结果突出了一种无差错绕过ssDNA中产生的脱氧尿苷的新机制，并表明在癌症基因组中观察到的APOBEC突变特征可能低估了这些酶诱导的基因组损伤。
