DNA-lesion mapping in mammalian cells

Abstract

Formation of DNA damage is a crucial event in carcinogenesis. Irreparable DNA lesions have the potential to cause mispairing during DNA replication, thereby giving rise to mutations. Critically important mutations in cancer-related genes, i.e., oncogenes and tumor suppressor genes, are key contributors to carcinogenesis. Theoretically, co-localization(s) of persistent DNA lesions and mutational hotspots in cancer-relevant genes can be used for causality inference. The inferred causality can be validated if a suspected carcinogen can similarly produce corresponding patterns of DNA damage and mutagenesis in vitro and/or in vivo. DNA-lesion footprinting (mapping) in conjunction with mutagenicity analysis is used for investigating cancer etiology. Ligation-mediated polymerase chain reaction (LM-PCR) is a versatile DNA-lesion footprinting technique, which enables sensitive and specific detection of DNA damage, at the level of nucleotide resolution, in genomic DNA. Here, we describe an updated protocol for LM-PCR analysis of the mammalian genome. This protocol can routinely be used for DNA-lesion footprinting of a variety of chemical and/or physical carcinogens in mammalian cells.

Figure 1. (a) Mutation spectrum and (b) codon distribution of the TP53 tumor suppressor gene in human non-melanoma skin tumors (basal cell and squamous cell carcinomas; n = 541)

Data were obtained from the TP53 mutation database of the International Agency for Research on Cancer (R12 version) (31). Ins: Insertions; Del: Deletions.

Figure 1. (a) Mutation spectrum and (b) codon distribution of the TP53 tumor suppressor gene in human non-melanoma skin tumors (basal cell and squamous cell carcinomas; n = 541)

Data were obtained from the TP53 mutation database of the International Agency for Research on Cancer (R12 version) (31). Ins: Insertions; Del: Deletions.

Figure 2. A schematic outline of the LM-PCR procedure

A lesion in the original DNA is indicated by ◆. P1, P2, and P3 indicate gene-specific Primers 1, 2, and 3, respectively.

Figure 3. LM-PCR with Maxam and Gilbert chemical reactions

Genomic DNA of Big Blue^® mouse embryonic fibroblasts was subjected to standard Maxam and Gilbert chemical reactions (20, 21), and subsequently LM-PCR was performed on the cII transgene. Individual Maxam/Gilbert sequencing ladders include “G”, “G + A”, “C”, and “C + T”. M = Molecular weight standard marker.