Dennen Matthew S, Kockler Zachary W, Roberts Steven A, Burkholder Adam B, Klimczak Leszek J, Gordenin Dmitry A
Genome Integrity & Structural Biology Laboratory, National Institute of Environmental Health Sciences, Durham, NC 27709.
Department of Microbiology and Molecular Genetics, University of Vermont Cancer Center, University of Vermont, Burlington, VT 05405.
bioRxiv. 2024 Jul 4:2024.06.27.601081. doi: 10.1101/2024.06.27.601081.
Human APOBEC single-strand (ss) specific DNA and RNA cytidine deaminases change cytosines to uracils and function in antiviral innate immunity, RNA editing, and can cause hypermutation in chromosomes. The resulting uracils can be directly replicated, resulting in C to T mutations, or uracil-DNA glycosylase can convert the uracils 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 depends 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 yeast and in strains carrying a hypomorph mutation in the large RPA subunit. We confirmed that the allele can facilitate mutagenesis by APOBECs. We also found that the 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 uracils in ssDNA from Ung1, thereby facilitating C to T mutagenesis through the accurate copying of uracils by replicative DNA polymerases. Unexpectedly, we also found that for uracils shielded from Ung1 by wild-type RPA the mutagenic outcome is reduced in the presence of translesion DNA polymerase zeta.
人类载脂蛋白B mRNA编辑酶催化多肽样单链（ss）特异性DNA和RNA胞嘧啶脱氨酶可将胞嘧啶转变为尿嘧啶，并在抗病毒固有免疫、RNA编辑中发挥作用，还可导致染色体超突变。由此产生的尿嘧啶可直接复制，导致C到T突变，或者尿嘧啶-DNA糖基化酶可将尿嘧啶转化为无碱基（AP）位点，然后经跨损伤DNA聚合酶将其固定为C到T或C到G突变。我们注意到，在酵母和人类癌症中，C到T和C到G突变的作用取决于载脂蛋白B mRNA编辑酶催化多肽样蛋白诱变的单链DNA的来源。由于真核基因组中的单链DNA很容易与复制蛋白A（RPA）结合，我们不禁要问，RPA是否会影响酵母中载脂蛋白B mRNA编辑酶催化多肽样蛋白诱导的突变谱。为此，我们在野生型酵母和携带大RPA亚基低表达突变的菌株中表达了人类载脂蛋白B mRNA编辑酶催化多肽样蛋白。我们证实，该等位基因可促进载脂蛋白B mRNA编辑酶催化多肽样蛋白的诱变作用。我们还发现，该突变改变了载脂蛋白B mRNA编辑酶催化多肽样蛋白3A在复制酵母中诱导的T到C和T到G突变的比例，使其类似于在酵母和癌症中长期存在的单链DNA中观察到的比例。我们提供的数据表明，RPA可能会保护单链DNA中载脂蛋白B mRNA编辑酶催化多肽样蛋白形成的尿嘧啶不被尿嘧啶-DNA糖基化酶1识别，从而通过复制性DNA聚合酶对尿嘧啶的准确复制促进C到T诱变。出乎意料的是，我们还发现，对于野生型RPA保护不被尿嘧啶-DNA糖基化酶1识别的尿嘧啶，在跨损伤DNA聚合酶ζ存在的情况下，诱变结果会降低。
