APOBEC-induced mutations in human cancers are strongly enriched on the lagging DNA strand during replication

Abstract

APOBEC3A and APOBEC3B, cytidine deaminases of the APOBEC family, are among the main factors causing mutations in human cancers. APOBEC deaminates cytosines in single-stranded DNA (ssDNA). A fraction of the APOBEC-induced mutations occur as clusters ("kataegis") in single-stranded DNA produced during repair of double-stranded breaks (DSBs). However, the properties of the remaining 87% of nonclustered APOBEC-induced mutations, the source and the genomic distribution of the ssDNA where they occur, are largely unknown. By analyzing genomic and exomic cancer databases, we show that >33% of dispersed APOBEC-induced mutations occur on the lagging strand during DNA replication, thus unraveling the major source of ssDNA targeted by APOBEC in cancer. Although methylated cytosine is generally more mutation-prone than nonmethylated cytosine, we report that methylation reduces the rate of APOBEC-induced mutations by a factor of roughly two. Finally, we show that in cancers with extensive APOBEC-induced mutagenesis, there is almost no increase in mutation rates in late replicating regions (contrary to other cancers). Because late-replicating regions are depleted in exons, this results in a 1.3-fold higher fraction of mutations residing within exons in such cancers. This study provides novel insight into the APOBEC-induced mutagenesis and describes the peculiarity of the mutational processes in cancers with the signature of APOBEC-induced mutations.

APOBEC3A damages the cellular genome during DNA 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.…”

“…4D). 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.…”

“…26 Seplyarskiy, et al recently provided a biological correlate to these in vitro data by identifying non-clustered APOBEC signature mutations in tumor genome sequences. 32 Thus, the degree of genomic damage caused by APOBEC3 enzymes is in part limited by the availability and length of exposed ssDNA. Consequently, identification of the cellular ssDNA substrates with which APO-BEC3 enzymes interact is critical to understanding their capacity to damage the cellular genome.…”

“…23 Additionally, expression of A3A and A3B in yeast can produce clusters of break-associated mutations indicative of deamination of resected DSBs. 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.…”