APOBEC3A damages the cellular genome during DNA replication

Abstract

The human APOBEC3 family of DNA-cytosine deaminases comprises 7 members (A3A-A3H) that act on single-stranded DNA (ssDNA). The APOBEC3 proteins function within the innate immune system by mutating DNA of viral genomes and retroelements to restrict infection and retrotransposition. Recent evidence suggests that APOBEC3 enzymes can also cause damage to the cellular genome. Mutational patterns consistent with APOBEC3 activity have been identified by bioinformatic analysis of tumor genome sequences. These mutational signatures include clusters of base substitutions that are proposed to occur due to APOBEC3 deamination. It has been suggested that transiently exposed ssDNA segments provide substrate for APOBEC3 deamination leading to mutation signatures within the genome. However, the mechanisms that produce single-stranded substrates for APOBEC3 deamination in mammalian cells have not been demonstrated. We investigated ssDNA at replication forks as a substrate for APOBEC3 deamination. We found that APOBEC3A (A3A) expression leads to DNA damage in replicating cells but this is reduced in quiescent cells. Upon A3A expression, cycling cells activate the DNA replication checkpoint and undergo cell cycle arrest. Additionally, we find that replication stress leaves cells vulnerable to A3A-induced DNA damage. We propose a model to explain A3A-induced damage to the cellular genome in which cytosine deamination at replication forks and other ssDNA substrates results in mutations and DNA breaks. This model highlights the risk of mutagenesis by A3A expression in replicating progenitor cells, and supports the emerging hypothesis that APOBEC3 enzymes contribute to genome instability in human tumors.

Functions and Malfunctions of Mammalian DNA-Cytosine Deaminases

“…3B). These may be the strand breaks observed by Green et al 279 when they expressed A3A in the S phase. Thus expression of APOBECs during replication should cause genome instability in addition to generating mutations.…”

“…The latter cells acquired more genomic uracils and strand breaks than the cells in G1. 279 Together, these studies implicate replication forks as targets for APOBEC3s. 280 …”

APOBEC3A Expression in Penile Squamous Cell Carcinoma

“…However, there is mounting evidence that APOBEC3 proteins are also able to function as endogenous mutators of host cellular DNA [5,6]. APOBEC3A has been shown to induce DNA double strand breaks and to activate the cellular DNA damage response [7][8][9]. The target DNA motif of most APOBEC3s is 5′-TC-3′ (mutated base underlined) and next generation sequencing has provided evidence for a mutational signature consistent with APOBEC3 editing in a number of cancers [10,11].…”

APOBEC3A/B-induced mutagenesis is responsible for 20% of heritable mutations in the TpCpW context

“…While deamination in the TpCpW context excludes some APOBECs from consideration, APOBEC1, APOBEC3A, APOBEC3B, APOBEC3F, and APOBEC3H (Taylor et al 2013;Kim et al 2014;Saraconi et al 2014) all are plausible suspects. Overexpression of APOBEC1, APOBEC3A, and APOBEC3B is associated with increased mutation rate in vertebrate cell lines, with mutations distributed all over the genome (Saraconi et al 2014;Akre et al 2016;Green et al 2016). Notably, however, APOBEC1 causes C→A mutations in experimental systems (Saraconi et al 2014), while the heritable replication asymmetry is largely restricted to C→T and C→G mutations (Table 1).…”

“…However, this APOBEC3H allele is barely stable and has only a weak effect even in specific essays , arguing against the role of APOBEC3H in the patterns observed in germline. For APOBEC3A and APOBEC3B, mutations are known to accumulate on the lagging strand (Haradhvala et al 2016;Hoopes et al 2016;Seplyarskiy et al 2016), and mutagenesis is associated with replication (Green et al 2016). Finally, APOBEC3A and APOBEC3B are major mutators in a broad range of cancer types (Alexandrov et al 2013;Burns et al 2013b;Roberts et al 2013).…”