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.

Spatiotemporal regulation of PCNA ubiquitination in damage tolerance pathways

“…Importantly, when BPDE-G lesions are proximally located in the two DNA strands, the fraction processed by HDR is reduced to approximately 18-27% (Izhar et al 2013;Cohen et al 2015). Single lesions integrated into one chromosome do not appear to induce the DNA damage response (Cohen et al 2015), indicating that DNA damage responses, such as cell cycle checkpoints, are not essential for pathway choice. Instead, it appears that intrinsic chemical properties of the DNA lesions regulate the choice of DDT pathway.…”

“…The HDR detected in this assay might differ from standard HDR, which requires a sister chromatid. The other method involves the integration of DNA fragments containing a defined lesion into the genome (Izhar et al 2013;Cohen et al 2015). The majority of TT-CPDs (99%) are processed by error-free TLS.…”

“…Pathway choice might be essentially independent of checkpoint responses because it is observed in the absence of a clear checkpoint response (Cohen et al 2015). Instead, it could be differentially regulated in the different stages of the cell cycle.…”

Error-prone bypass of DNA lesions during lagging strand replication is a common source of germline and cancer mutations

“…Experiments in well-controlled genetic systems and in model organisms have uncovered that DNA polymerases make errors and resulting mismatches become mutations 1 . An alternative mechanism of mutagenesis due to misrepaired DNA damage or DNA damage bypassed by translesion (TLS) polymerases has been extensively studied in experimental systems exposed to exogenous mutagens 2,3 . Although these studies do shed light on the mechanistic details of mutagenesis, well-controlled experimental systems provide little information on the relative contributions of these mechanisms to naturally occurring human mutations.…”

DNA sequence context greatly affects the accuracy of bypass across an ultraviolet light 6-4 photoproduct in mammalian cells

“…Translesion DNA synthesis (TLS) utilizes specialized low-fidelity DNA polymerases to bypass the lesions, whereas homology-dependent repair (HDR, also termed postreplication repair, damage avoidance, or template switch) utilizes the homologous sister chromatid as a DNA sequence source for bypassing the lesion [2–4]. While TLS is inherently inaccurate due to the miscoding of most DNA lesions [5,6], HDR is fundamentally accurate [7–10]. Remarkably, despite the great heterogeneity of DNA lesions, TLS often incorporates the correct nucleotide(s) opposite it, leading to accurate TLS [5].…”