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.

Keywords: APOBEC3A; APOBEC3B; cancer genome mutations; kataegis; uracil-DNA glycosylase.

Fig. 1.

Distribution and sequence context of C:G to T:A mutations. (A) Distribution of mutations. The positions of C:G to T:A mutations were plotted using the Graph Prism 6 for Mac software and are shown using the perpendicularity symbol (⊥); the strains in which the mutations occurred are indicated on the left. A straight line at the bottom is used to represent the E. coli genome, and downward arrows mark the positions of the replication origin (Ori) and terminus (Ter). (B) Sequence context of mutations. The three nucleotides on either side of the C:G pair that was mutated to T:A were used to create LOGOS plots using enoLOGOS software (63).

Fig. S1.

Sequence context of cytosines mutated to thymine in WT strain without a plasmid and in ung mutant strain with the plasmid pA3G-CTDmut. The three nucleotides on either side of the C:G pair that was mutated to T:A were used to create LOGOS plots using enoLOGOS software (63). Analysis of mutations in the WT strain (A) and the ung mutant strain containing plasmid pA3G-CTDmut (B).

Fig. S2.

Purple rightward arrows (→) show the overall direction of replication. Origin (ORI) and termination (TER) of replication are shown. Replication starts at ORI and proceeds in both clockwise (purple, Replichore 1) and anticlockwise (green, Replichore 2) fashion. The start of the E. coli sequence is shown at the top, and the strand directionality is shown at the ORI. The lagging and leading strand templates (LGST and LDST, respectively) are labeled for each replichore. Representative deaminations of cytosines in the LGST and LDST of each replichore are shown.

Fig. 2.

Consequences of cytosine deaminations at the replication fork. Deamination of three cytosines and the consequences of processing of the resulting uracils through replication or repair pathways are shown. Copying of the LGST is discontinuous, and two Okazaki fragments are shown. The open arrowhead represents the helicase DnaB and is pointed in the overall direction of replication. Class 1 and class 2 refer to two classes of uracils generated through cytosine deamination and have different biochemical consequences. The class 2 uracils are likely to be replaced with cytosines through BER, whereas the class 1 uracils lead to C:G to T:A mutations or double-strand (DS) breaks. The latter may be repaired through recombinational repair or result in the collapse of the replication fork.