Imaging cellular responses to antigen tagged DNA damage

Abstract

Repair pathways of covalent DNA damage are understood in considerable detail due to decades of brilliant biochemical studies by many investigators. An important feature of these experiments is the defined adduct location on oligonucleotide or plasmid substrates that are incubated with purified proteins or cell free extracts. With some exceptions, this certainty is lost when the inquiry shifts to the response of living mammalian cells to the same adducts in genomic DNA. This reflects the limitation of assays, such as those based on immunofluorescence, that are widely used to follow responding proteins in cells exposed to a DNA reactive compound. The lack of effective reagents for adduct detection means that the proximity between responding proteins and an adduct must be assumed. Since these assumptions can be incorrect, models based on in vitro systems may fail to account for observations made in vivo. Here we discuss the use of a detection tag to address the problem of lesion location, as illustrated by our recent work on replication dependent and independent responses to interstrand crosslinks.

Keywords: Antigen tag; DNA damage response; Interstrand crosslink; Replication independent repair; Replication stress.

Fig.1.

The structure of Digoxigenin tagged psoralen

Fig. 2.. Interpretation of patterns of tracts with encounters with an ICL.

A. Experimental scheme and representative images of the patterns observed. The replication restart pattern is termed fork traverse. B. Scheme summarizing the possible outcomes observed when the replication fork encounters a Dig-TMP/UVA induced ICL. The pie chart shows the relative frequency of the major patterns in wt cells. FT, replication fork traverse, SF, single fork, DF, double fork.

Fig. 3.. Strategies designed to study replication independent ICL repair employing laser localized tagged ICLs.

A. Laser-localized ICLs combined with immunofluorescence to study DDR activation by, and repair of, laser localized ICLs. γ-H2AX and the FAN1 nuclease appear in ICL stripes. Monitoring the Dig tag reveals repair of the ICL and the greater persistence of γ-H2AX. B. Laser-localized ICL induction and proximity ligation assay (PLA) to distinguish DDR proteins that are proximal or distal to the tagged ICL.