WRNIP1 Is Recruited to DNA Interstrand Crosslinks and Promotes Repair

Abstract

Summary The Fanconi anemia (FA) pathway repairs DNA interstrand crosslinks (ICLs). Many FA proteins are recruited to ICLs in a timely fashion so that coordinated repair can occur. However, the mechanism of this process is poorly understood. Here, we report the purification of a FANCD2-containing protein complex with multiple subunits, including WRNIP1. Using live-cell imaging, we show that WRNIP1 is recruited to ICLs quickly after their appearance, promoting repair. The observed recruitment facilitates subsequent recruitment of the FANCD2/FANCI complex. Depletion of WRNIP1 sensitizes cells to ICL-forming drugs. We find that ubiquitination of WRNIP1 and the activity of its UBZ domain are required to facilitate recruitment of FANCD2/FANCI and promote repair. Altogether, we describe a mechanism by which WRNIP1 is recruited rapidly to ICLs, resulting in chromatin loading of the FANCD2/FANCI complex in an unusual process entailing ubiquitination of WRNIP1 and the activity of its integral UBZ domain.

Purification of DNA repair protein complexes from mammalian cells

“…The clean mock purification and the reproducibility of purified complexes indicate the robustness of the protocol. Parts of this figure (marker lane and lanes 1 and 3) are identical to previously published data, and are reprinted with permission ( Socha et al., 2020 ). …”

“…In this protocol, we purify FANCD2 sequentially via the FLAG and HA tags engineered at its N-terminus. Though using this protocol, we typically observe negligible level of contaminants ( Socha et al., 2020 ), we recommend performing a parallel “mock” purification from cells not expressing a FLAG-HA-tagged protein, to assess the level of background binding proteins.…”

“…An example of this is shown in Figure 3 , which show two independent purifications of FANCD2 complexes. One of the two purifications is identical to a previously published purification ( Socha et al., 2020 ). The gel image is shown again here, side-by-side with an independent purification, as an example of how robust and reproducible the method is.…”

“…This protocol can be applied to uncover the functions and mechanism of DNA repair pathways. For complete information on the use and execution of this protocol, please refer to Socha et al. (2020) .…”

“…For complete information on the use and execution of this protocol, please refer to Socha et al. (2020) .…”