Search

Considerable insight is present into the cellular response to double strand breaks (DSBs), induced by nucleases, radiation, and other DNA breakers. In part, this reflects the availability of methods for the identification of break sites, and characterization of factors recruited to DSBs at those sequences. However, DSBs also appear as intermediates during the processing of DNA adducts formed by compounds that do not directly cause breaks, and do not react at specific sequence sites. Consequently, for most of these agents, technologies that permit the analysis of binding interactions with response factors and repair proteins are unknown. For example, DNA interstrand crosslinks (ICLs) can provoke breaks following replication fork encounters. Although formed by drugs widely used as cancer chemotherapeutics, there has been no methodology for monitoring their interactions with replication proteins. Here, we describe our strategy for following the cellular response to fork collisions with these challenging adducts. We linked a steroid antigen to psoralen, which forms photoactivation dependent ICLs in nuclei of living cells. The ICLs were visualized by immunofluorescence against the antigen tag. The tag can also be a partner in the Proximity Ligation Assay (PLA) which reports the close association of two antigens. The PLA was exploited to distinguish proteins that were closely associated with the tagged ICLs from those that were not. It was possible to define replisome proteins that were retained after encounters with ICLs and identify others that were lost. This approach is applicable to any structure or DNA adduct that can be detected immunologically.

Replication forks encounter numerous challenges as they move through eu- and hetero-chromatin during S phase in mammalian cells. These include a variety of impediments to the unwinding of DNA by the replicative helicase such as alternate DNA structures, transcription complexes and R-loops, DNA–protein complexes, and DNA chemical adducts. Much of our knowledge of these events is based on analysis of markers of the replication stress and DNA Damage Response that follow stalling of replisomes. To examine consequences for the replisomes more directly, we developed an approach for imaging collisions of replication forks with the potent block presented by an interstrand crosslink (ICL). The strategy is based on the visualization on DNA fibers of the encounter of replication tracts and an antigen tagged ICL. Our studies revealed an unexpected restart of DNA synthesis past an intact ICL. In addition, and also unexpected, we found two distinct versions of the replisome, one biased toward euchromatin and the other more prominent in heterochromatin. Here, we present details of our experimental procedures that led to these observations.

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live cells. The DDR to helix distorting covalent DNA modifications, including interstrand DNA crosslinks (ICLs), is not as well defined. We have studied the DDR stimulated by ICLs, localized by laser photoactivation of immunotagged psoralens, in the nuclei of live cells. In order to address fundamental questions about adduct distribution and replication fork encounters, we combined laser localization with two other technologies. DNA fibers are often used to display the progress of replication forks by immunofluorescence of nucleoside analogues incorporated during short pulses. Immunoquantum dots have been widely employed for single molecule imaging. In the new approach, DNA fibers from cells carrying laser localized ICLs are spread onto microscope slides. The tagged ICLs are displayed with immunoquantum dots and the inter-lesion distances determined. Replication fork collisions with ICLs can be visualized and different encounter patterns identified and quantitated.

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. : Replication of the mammalian genome is driven by the replisome complex, which unwinds DNA and must overcome many impediments. Zhang et al. find that the encounter of the CMG with a strong block triggers a change in replisome composition that is important for restart of replication past the obstruction. Keywords: replication traverse, interstrand crosslink, ICL, ATR, FANCM, FANCD2, CMG, GINS

DNA interstrand crosslinks (ICLs) block unwinding of the double helix, and have always been regarded as major challenges to replication and transcription. Compounds that form these lesions are very toxic and are frequently used in cancer chemotherapy. We have developed two strategies, both based on immunofluorescence, for studying cellular responses to ICLs. The basis of each is psoralen, a photoactive (by long wave ultraviolet light, UVA) DNA crosslinking agent, to which we have linked an antigen tag. In the one approach, we have taken advantage of DNA fiber and immunoquantum dot technologies for visualizing the encounter of replication forks with ICLs induced by exposure to UVA lamps. In the other, psoralen ICLs are introduced into nuclei in live cells in regions of interest (ROI) defined by a UVA laser. The antigen tag can be displayed by conventional immunofluorescence, as can the recruitment and accumulation of DNA Damage Response (DDR) proteins to the laser localized ICLs. However, substantial difference between the technologies creates considerable uncertainty as to whether conclusions from one approach are applicable to those of the other. In this report we have employed the fiber/quantum dot methodology to determine lesion density and spacing on individual DNA molecules carrying laser localized ICLs. We have performed the same measurements on DNA fibers with ICLs induced by exposure of psoralen to UVA lamps. Remarkably, we find little difference in the adduct distribution on fibers prepared from cells exposed to the different treatment protocols.. Furthermore, there is considerable similarity in patterns of replication in the vicinity of the ICLs introduced by the two techniques