Replication-coupled DNA interstrand cross-link repair in Xenopus egg extracts

Abstract

Interstrand cross-links (ICL) are one of the most hazardous types of DNA damage as they form a roadblock to all processes that involve strand separation. Repair of these lesions involves several different DNA repair pathways, but the molecular mechanism is unclear. Here we describe a system that allows the examination of ICL repair, via a physiological mechanism, in vitro. This system, which uses Xenopus egg extracts in combination with a DNA template that contains a site-specific ICL, represents a unique tool to study the molecular mechanism of ICL repair.

Fig 1

Schematic Overview of DNA repair intermediates and products generated by the various assays described in this chapter. (a) Cartoon of pICL showing the restriction sites and intermediates formed during repair. (b) DNA products analyzed on sequencing gels in the lesion bypass assay (Subheading 3.3). Dark grey and red strands are visible on the gel in Fig. 2. Lesion bypass of the rightward moving fork (red strands) can be followed at single nucleotide resolution (see Fig. 2). (c) Products analyzed under denaturing and native conditions in the incision and repair assays respectively (Subheadings 3.4 and 3.5).

Fig. 2

Analysis of lesion bypass during ICL repair (Subheading 3.3). AflIII digested repair intermediates are separated on a sequencing gel. Leading and lagging strands of the leftward moving fork, leading strands of the rightward moving fork, and lesion bypass product (‘Extension product’) are indicated on the left panel. Right panel shows an enlargement of the rightward moving leading strand on which −20 to −40, −1, 0, and extension products are indicated.

Fig. 3

Examination of dual incisions during ICL repair. HincII digested repair intermediates are separated on a denaturing gel and visualized using Southern blotting (land 5–14) (Subheading 3.4). Undigested and HincII and/or SapI digested pControl and pICL are shown in lane 1–4 and serve as size markers. X-structures, linears, and 2.3/3.3 arm fragments are indicated. pQuant is visible due to some background reactivity with the probe.

Fig. 4

Determination of ICL repair efficiency. (a) Analysis of HincII (lane 1–10) and HincII/SapI (lane 11–20) digested repair intermediates on a native agarose gel (Subheading 3.5). (b) Calculated repair efficiency plotted against time.