Formaldehyde-induced mutagenesis in Saccharomyces cerevisiae: molecular properties and the roles of repair and bypass systems

Abstract

Although DNA-protein cross-links (DPCs) pose a significant threat to genome stability, they remain a poorly understood class of DNA lesions. To define genetic impacts of DPCs on eukaryotic cells in molecular terms, we used a sensitive Saccharomyces cerevisiae frameshift-detection assay to analyze mutagenesis by formaldehyde (HCHO), and its response to nucleotide excision repair (NER) and translesion DNA synthesis (TLS). Brief exposure to HCHO was mutagenic for NER-defective rad14 strains but not for a corresponding RAD14 strain, nor for a rad14 strain lacking both Polζ and Polη TLS polymerases. This confirmed that HCHO-generated DNA lesions can trigger error-prone TLS and are substrates for the NER pathway. Sequencing revealed that HCHO-induced single-base-pair insertions occurred primarily at one hotspot; most of these insertions were also complex, changing an additional base-pair nearby. Most of the HCHO-induced mutations required both Polζ and Polη, providing a striking example of cooperativity between these two TLS polymerases during bypass of a DNA lesion formed in vivo. The similar molecular properties of HCHO-induced and spontaneous complex +1 insertions detected by this system suggest that DPCs which form in vivo during normal metabolism may contribute characteristic events to the spectra of spontaneous mutations in NER-deficient cells.

Figure 1

Effect of formaldehyde treatment and NER status on frameshift mutagenesis. The nucleotide sequence represents the reversion window of the lys2ΔA746,NR allele, in which the position of the deleted A is marked by a hyphen. Each event affecting fewer than 12 bp is represented individually; larger events are summarized under captions. Small insertions and deletions are indicated above and below the sequence, respectively; complex insertions are designated “cins”. HS3 is highlighted yellow; type 1 and 2 events are in red and type 3 events are in green.

Figure 2

Effect of TLS polymerases on formaldehyde-induced frameshift mutagenesis. See Figure 1 legend for details.

Figure 3

Proposed mechanisms for formaldehyde-induced mutagenesis at the hotspot “HS3”. The three schemes summarize events catalyzed by Polζ and Polη that can generate the observed HCHO-induced, Rev3-dependent complex insertions. For simplicity the initiating DPC (indicated by the yellow star) is depicted at the guanine between the 3T and 3A runs, but other positions are possible. The base inserted opposite the initiating DPC is in red.