Increased mutagenesis and unique mutation signature associated with mitotic gene conversion

Abstract

To examine the fidelity of DNA synthesis during double-strand break (DSB) repair in Saccharomyces cerevisiae we studied gene conversion in which both strands of DNA are newly synthesized. The mutation rate increases up to 1400 times over spontaneous events, with a significantly different mutation signature. Especially prominent are microhomology-mediated template switches. Recombination-induced mutations are largely independent of mismatch repair, by DNA polymerases Polzeta, Poleta, and Pol32, but result from errors made by Poldelta and Polepsilon. These observations suggest that increased DSB frequencies in oncogene-activated mammalian cells may also increase the probability of acquiring mutations required for transition to a cancerous state.

Figure 1

(A) MATα switching occurs via SDSA (4). DNA synthesis beginning in HMR-Z copies Y sequences and progresses into HMR-X. The nascent strand dissociates and anneals to MAT-X sequences on the other side of the DSB. Nonhomologous sequences are clipped off and second strand synthesis initiates using the first strand as a template. Nonprocessive DNA synthesis results in premature dissociation from HMR, which can result in error-prone reannealing to the HMR donor template or to other ectopic templates. To complete the GC, template switch events must dissociate again and reanneal to HMR. (B) GC-associated quasi-palindrome template switch at position 584-585 within the Kl-URA3 ORF. (C) GC-associated ectopic template switch into Sc-ura3-52 and then back to hmr::Kl-URA3 in which a frameshift is introduced due to misalignment at a shared 5-bp sequence. Underlined sequences indicate Kl-URA3 microhomology (nts 423-427).

Figure 2

GC-associated Kl-URA3 mutant spectrum. Single letters above the Kl-URA3 ORF indicate BPSs. Black boxes with white text indicate nonsense mutations. Insertions and deletions are shown below the ORF; insertions are green and deletions are red. A large deletion occurring between direct repeats is indicated with repeats underlined in red. Blue lines below the ORF mark two ectopic events where Kl-URA3 sequences were replaced by Sc-ura3-52 sequences; double blue lines depict microhomology junctions. The template switch marked as (2) contained two independent jumps to Sc-ura3-52, one of which contained a frameshift. Quasi-palindrome mediated template switch mutations are within blue boxes (Figs. 1C & S3 depict these mechanisms). Recurrent mutations are indicated by an “X” followed by the number of times that mutation was observed.