DNA lesion identity drives choice of damage tolerance pathway in murine cell chromosomes

Abstract

DNA-damage tolerance (DDT) via translesion DNA synthesis (TLS) or homology-dependent repair (HDR) functions to bypass DNA lesions encountered during replication, and is critical for maintaining genome stability. Here, we present piggyBlock, a new chromosomal assay that, using piggyBac transposition of DNA containing a known lesion, measures the division of labor between the two DDT pathways. We show that in the absence of DNA damage response, tolerance of the most common sunlight-induced DNA lesion, TT-CPD, is achieved by TLS in mouse embryo fibroblasts. Meanwhile, BP-G, a major smoke-induced DNA lesion, is bypassed primarily by HDR, providing the first evidence for this mechanism being the main tolerance pathway for a biologically important lesion in a mammalian genome. We also show that, far from being a last-resort strategy as it is sometimes portrayed, TLS operates alongside nucleotide excision repair, handling 40% of TT-CPDs in repair-proficient cells. Finally, DDT acts in mouse embryonic stem cells, exhibiting the same pattern—mutagenic TLS included—despite the risk of propagating mutations along all cell lineages. The new method highlights the importance of HDR, and provides an effective tool for studying DDT in mammalian cells.

Figure 1.

The piggyBlock assay system. (A) A cassette consisting of DNA lesion(s) and puromycin selection marker is transposed from the piggyBlock constructed plasmid into a chromosomal locus by piggyBac transposase expressed from a co-transfected helper plasmid. (B) Local sequences of double-stranded lesion oligos ligated into the piggyBlock vector to form the constructed lesion plasmid. A star represents a benzo[a]pyrene adduct and a right angle above two pyrimidines represents dimerization. (C) Experiment timeline: constructed piggyBlock lesion plasmid and helper plasmid were co-transfected into cells. 24–48 h later, puromycin selection was administered. Cells were maintained under selection for 4–10 days, until colonies formed. Individual colonies were then transferred into wells of 96-well culture dishes and cultivated to full confluence (2–5 days). Colonies were harvested, chromosomal DNA was isolated and sequence analysis was performed. (D) Chromosomal integration loci identified by iPCR.

Figure 2.

Lesion bypass pathways and corresponding sequence signatures. A non-complementary nucleotide that is rarely inserted by TLS was engineered opposite the damaged base(s). During the first-replication post-transfection, bypass by accurate TLS inserts the correct complementary base (C) opposite the damaged template base (BP-G; marked by a star), while replication on the opposite strand in the same cell places A opposite the template T. Further rounds of replication result in a mosaic colony, and the lesion position is visualized as a double peak in the sequence file. Bypass by HDR in the first replication places T in the same sequence position, that is inserted opposite the A in the nascent sister chromatid that serves as alternative template. This is visualized in the sequence file as a single peak, corresponding to the intact strand sequence.

Figure 3.

DNA sequence outcome of piggyBlock TLS/HDR experiments in MEF. (A) TT-CPD single lesion, n = 168. (B) BP-G single lesion, n = 242. (C) TT-CPD dual lesion, n = 240. (D) BP-G dual lesion, n = 159.

Figure 4.

DNA sequence outcome of piggyBlock TLS/HDR and TLS/NER experiments in mES cells. (A) BP-G in NER-deficient mESC, n = 398. (B) TT-CPD in NER-deficient mESC, n = 146. (C) TT-CPD in NER-proficient mESC, n = 195.