Remodeling of Interstrand Crosslink Proximal Replisomes Is Dependent on ATR, FANCM, and FANCD2

Abstract

SUMMARY Eukaryotic replisomes are driven by the mini chromosome maintenance (MCM [M]) helicase complex, an offset ring locked around the template for leading strand synthesis by CDC45 (C) and GINS (G) proteins. Although the CDC45 MCM GINS (CMG) structure implies that interstrand crosslinks (ICLs) are absolute blocks to replisomes, recent studies indicate that cells can restart DNA synthesis on the side of the ICL distal to the initial encounter. Here, we report that restart requires ATR and is promoted by FANCD2 and phosphorylated FANCM. Following introduction of genomic ICLs and dependent on ATR and FANCD2 but not on the Fanconi anemia core proteins or FAAP24, FANCM binds the replisome complex, with concomitant release of the GINS proteins. In situ analysis of replisomes proximal to ICLs confirms the ATR-dependent release of GINS proteins while CDC45 is retained on the remodeled replisome. The results demonstrate the plasticity of CMG composition in response to replication stress.

DONSON and FANCM associate with different replisomes distinguished by replication timing and chromatin domain

“…These data demonstrated the association of DONSON with replisomes in cells with or without TMP/UVA treatment. Furthermore, they distinguished DONSON from FANCM, which, as shown previously, was not in complex with GINS proteins in either condition 29 .…”

“…DONSON was also complexed with CDC45 and the GINS proteins indicating association with the helicase functional form of the replisome ( Supplementary Fig. 1f ), in contrast to replisomes bound by FANCM 29 . Proximity ligation assays (PLA) confirmed these interactions ( Supplementary Fig.…”

“…FANCM was recruited to ICL proximal replisomes which were marked by phosphorylation of MCM2 by ATR. Furthermore, the association with FANCM was accompanied by remodeling of replisomes characterized by the loss of the GINS complex 29 .…”

Identification of MCM8IP, an interactor of MCM8-9 and RPA1 that promotes homologous recombination and DNA synthesis in response to DNA damage

“…However, a mechanism involving traverse of an ICL by the replisome without significantly compromising fork progression has recently been described 56 . While the exact details of this process need to be elucidated, ICL traverse involves remodeling of the CDC45-MCM2-7-GINS (CMG) helicase 59 . It has been suggested that FANCM, BLM 60 , or an as yet unidentified helicase(s) facilitates replisome traverse in part by generating ssDNA beyond the ICL, which allows an open replicative helicase complex to re-encircle DNA after translocation 37 .…”

The GINS complex is required for the survival of rapidly proliferating retinal and tectal progenitor cells during zebrafish development

“…Prior to this, the MCM-complex may be able to perform its helicase function in the absence of GINS. Human dermal fibroblasts lacking GINS2 are also able to undergo mitosis, albeit at a slower rate, providing evidence that GINS-independent proliferation can also occur in mammalian cells (Barkley et al, 2009), and recent results suggest that other proteins, such as Fancm could substitute for GINS function under specific circumstances (Huang et al, 2019). It will be important to elucidate the identity of factors that can perform GINSlike function in young zebrafish embryos as they might be important, yet unrecognized, conditional components of the vertebrate replisome.…”