Genome Integrity & Structural Biology Laboratory, National Institute of Environmental Health Sciences, Durham, NC 27709, USA.
Department of Microbiology and Molecular Genetics, University of Vermont Cancer Center, University of Vermont, Burlington, VT 05405, USA.
G3 (Bethesda). 2024 Oct 7;14(10). doi: 10.1093/g3journal/jkae196.
Human APOBEC single-strand (ss) specific DNA and RNA cytidine deaminases change cytosines to uracils (U's) and function in antiviral innate immunity and RNA editing and can cause hypermutation in chromosomes. The resulting U's can be directly replicated, resulting in C to T mutations, or U-DNA glycosylase can convert the U's to abasic (AP) sites which are then fixed as C to T or C to G mutations by translesion DNA polymerases. We noticed that in yeast and in human cancers, contributions of C to T and C to G mutations depend on the origin of ssDNA mutagenized by APOBECs. Since ssDNA in eukaryotic genomes readily binds to replication protein A (RPA) we asked if RPA could affect APOBEC-induced mutation spectrum in yeast. For that purpose, we expressed human APOBECs in the wild-type (WT) yeast and in strains carrying a hypomorph mutation rfa1-t33 in the large RPA subunit. We confirmed that the rfa1-t33 allele can facilitate mutagenesis by APOBECs. We also found that the rfa1-t33 mutation changed the ratio of APOBEC3A-induced T to C and T to G mutations in replicating yeast to resemble a ratio observed in long persistent ssDNA in yeast and in cancers. We present the data suggesting that RPA may shield APOBEC formed U's in ssDNA from Ung1, thereby facilitating C to T mutagenesis through the accurate copying of U's by replicative DNA polymerases. Unexpectedly, we also found that for U's shielded from Ung1 by WT RPA, the mutagenic outcome is reduced in the presence of translesion DNA polymerase zeta.
人类 APOBEC 单链 (ss) 特异性 DNA 和 RNA 胞嘧啶脱氨酶将胞嘧啶转化为尿嘧啶 (U)，并在抗病毒先天免疫和 RNA 编辑中发挥作用，可导致染色体发生超突变。产生的 U 可直接复制，导致 C 突变为 T，或 U-DNA 糖苷酶可将 U 转化为无碱基 (AP) 位点，然后由跨损伤 DNA 聚合酶将其固定为 C 突变为 T 或 C 突变为 G。我们注意到，在酵母和人类癌症中，C 突变为 T 和 C 突变为 G 的突变取决于 APOBEC 诱变的 ssDNA 的来源。由于真核基因组中的 ssDNA 容易与复制蛋白 A (RPA) 结合，我们想知道 RPA 是否会影响酵母中 APOBEC 诱导的突变谱。为此，我们在野生型 (WT) 酵母和携带大 RPA 亚基 rfa1-t33 部分缺陷突变的菌株中表达了人类 APOBEC。我们证实 rfa1-t33 等位基因可以促进 APOBEC 的突变。我们还发现 rfa1-t33 突变改变了复制酵母中 APOBEC3A 诱导的 T 突变为 C 和 T 突变为 G 的比例，类似于在酵母和癌症中长期存在的 ssDNA 中观察到的比例。我们提出的数据表明，RPA 可能会保护 ssDNA 中由 APOBEC 形成的 U 免受 Ung1 的影响，从而通过复制 DNA 聚合酶准确复制 U 来促进 C 突变为 T 突变。出乎意料的是，我们还发现，对于被 WT RPA 屏蔽的 Ung1 的 U，在存在跨损伤 DNA 聚合酶 ζ 的情况下，突变的结果会减少。
