Strand-biased cytosine deamination at the replication fork causes cytosine to thymine mutations in Escherichia coli

Abstract

The rate of cytosine deamination is much higher in single-stranded DNA (ssDNA) than in double-stranded DNA, and copying the resulting uracils causes C to T mutations. To study this phenomenon, the catalytic domain of APOBEC3G (A3G-CTD), an ssDNA-specific cytosine deaminase, was expressed in an Escherichia coli strain defective in uracil repair (ung mutant), and the mutations that accumulated over thousands of generations were determined by whole-genome sequencing. C:G to T:A transitions dominated, with significantly more cytosines mutated to thymine in the lagging-strand template (LGST) than in the leading-strand template (LDST). This strand bias was present in both repair-defective and repair-proficient cells and was strongest and highly significant in cells expressing A3G-CTD. These results show that the LGST is accessible to cellular cytosine deaminating agents, explains the well-known GC skew in microbial genomes, and suggests the APOBEC3 family of mutators may target the LGST in the human genome.uracil-DNA glycosylase | APOBEC3A | APOBEC3B | kataegis | cancer genome mutations P airing of complementary DNA strands protects the DNA bases against modification by a number of hydrolytic, oxidizing, and alkylating chemicals (1-4). For example, water reacts with cytosine, creating uracil, and the rate of this reaction in single-stranded DNA (ssDNA) is more than 100-fold the rate in double-stranded DNA [dsDNA (5-7)]. Uracil-DNA glycosylase (Ung) excises uracils created by cytosine deamination in both ssDNA and dsDNA, resulting in abasic (AP) sites. In dsDNA, the AP sites are replaced with cytosines as a result of copying of the guanine in the complementary strand during repair by the base-excision repair (BER) pathway (8). In contrast, cytosine deaminations occurring in ssDNA are problematic because the complementary strand is not available to the BER pathway. Uracils that escape repair create C:G to T:A mutations, and incomplete repair of uracils can result in persistent AP sites and strand breaks that can destabilize the genome. Hence, identifying ssDNA regions that are susceptible to damage will increase our understanding of causes of mutations and genome instability.The AID/APOBEC (activation-induced deaminase/apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) family of DNA-cytosine deaminases are specific for ssDNA (9, 10). They are found only in vertebrates and are good probes of ssDNA in cells because of their relatively small size (about 190-amino acid catalytic domain). They are active in heterologous hosts such as Escherichia coli (11, 12) and yeast (13-15) and cause mutations in the same sequence context as in their known targets, such as Ig genes and the DNA copy of HIV-1 genome (11,16,17). In particular, the catalytic domain of human APOBEC3G (A3G-CTD) was expressed in an engineered yeast strain lacking the UNG gene and was shown to target ssDNA generated through aberrant resection of telomeric ends (13).To similarly probe ssDNA in E. coli, we expressed A3G-CTD on a plasmid (pA3G-CTD) in...

APOBEC3A damages the cellular genome during DNA replication

“…22 The ssDNA at replication forks has been suggested as an additional substrate that is susceptible to APOBEC3 deamination based on recent data from genome sequencing analyses 32,33 and model organism systems. 30,31 These studies point to the potential for APOBEC3 enzymes to deaminate various cellular substrates, however the capacity for A3A to damage ssDNA during replication has not been previously demonstrated in mammalian cells.…”

“…While this manuscript was under review, several groups published complementary data suggesting APOBEC3 deamination of ssDNA at replication forks in various computational examinations of tumor genome sequences 32,33 and model organism evaluations utilizing ectopic APO-BEC3 expression. 30,31 Notably, Hoopes, et al demonstrated preferential deamination of the lagging strand template in replicating yeast exposed to human A3A and A3B. 31 These findings were echoed in a genome sequence analysis of E. coli by Bhagwat, et al following exposure to the C-terminal deaminase domain of A3G over many generations of replication.…”

“…31 These findings were echoed in a genome sequence analysis of E. coli by Bhagwat, et al following exposure to the C-terminal deaminase domain of A3G over many generations of replication. 30 Additionally, analysis of nearly 4000 whole-genome and wholeexome sequenced cancers by Seplyarskiy, et al indicated enrichment of APOBEC3 signature mutations on lagging strand templates. 32 Together with our findings, this growing body of data reveals the susceptibility of ssDNA exposed during replication to mutagenesis by APOBEC3 enzymes.…”

“…This has been demonstrated by ectopic expression of human APOBEC3 enzymes in model organisms as well as analysis of cancer genomes. [30][31][32][33] Although these studies suggest ssDNA at replication forks is vulnerable to deamination by APOBECs, this has not been established experimentally in mammalian cells.…”

Functional Upregulation of the DNA Cytosine Deaminase APOBEC3B by Polyomaviruses

“…6E). The latter is supported by several recent studies investigating the nature of APOBEC-mediated mutations in various cancers (36)(37)(38)(39). However, considering the gene organization of the BKPyV genome and the bidirectional nature of its genome replication, we cannot at this time definitively determine whether transcription or lagging-strand DNA replication predisposes to more viral genomic mutation.…”