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FANCJ, a DNA helicase and interacting partner of the tumor suppressor BRCA1, is crucial for the repair of DNA interstrand crosslinks (ICL), a highly toxic lesion that leads to chromosomal instability and perturbs normal transcription. In diploid cells, FANCJ is believed to operate in homologous recombination (HR) repair of DNA double-strand breaks (DSB); however, its precise role and molecular mechanism is poorly understood. Moreover, compensatory mechanisms of ICL resistance when FANCJ is deficient have not been explored. In this work, we conducted a siRNA screen to identify genes of the DNA damage response/DNA repair regime that when acutely depleted sensitize FANCJ CRISPR knockout cells to a low concentration of the DNA cross-linking agent mitomycin C (MMC). One of the top hits from the screen was RAP80, a protein that recruits repair machinery to broken DNA ends and regulates DNA end-processing. Concomitant loss of FANCJ and RAP80 not only accentuates DNA damage levels in human cells but also adversely affects the cell cycle checkpoint, resulting in profound chromosomal instability. Genetic complementation experiments demonstrated that both FANCJ’s catalytic activity and interaction with BRCA1 are important for ICL resistance when RAP80 is deficient. The elevated RPA and RAD51 foci in cells co-deficient of FANCJ and RAP80 exposed to MMC are attributed to single-stranded DNA created by Mre11 and CtIP nucleases. Altogether, our cell-based findings together with biochemical studies suggest a critical function of FANCJ to suppress incompletely processed and toxic joint DNA molecules during repair of ICL-induced DNA damage.

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.

Purpose: Somatic inactivating mutations in ARID1A, a component of the SWI/SNF chromatin remodeling complex, are detected in various types of human malignancies. Loss of ARID1A compromises DNA damage repair. The induced DNA damage burden may increase reliance on PARP-dependent DNA repair of cancer cells to maintain genome integrity and render susceptibility to PARP inhibitor therapy. Experimental Design: Isogenic ARID1A−/− and wild-type cell lines were used for assessing DNA damage response, DNA compactness, and profiling global serine/threonine phosphoproteomic in vivo. A panel of inhibitors targeting DNA repair pathways was screened for a synergistic antitumor effect with irradiation in ARID1A−/− tumors. Results: ARID1A-deficient endometrial cells exhibit sustained levels in DNA damage response, a result further supported by in vivo phosphoproteomic analysis. Our results show that ARID1A is essential for establishing an open chromatin state upon DNA damage, a process required for recruitment of 53BP1 and RIF1, key mediators of non-homologous end-joining (NHEJ) machinery, to DNA lesions. The inability of ARID1A−/− cells to mount NHEJ repair results in a partial cytotoxic response to radiation. Small-molecule compound screens revealed that PARP inhibitors act synergistically with radiation to potentiate cytotoxicity in ARID1A−/− cells. Combination treatment with low-dose radiation and olaparib greatly improved antitumor efficacy, resulting in long-term remission in mice bearing ARID1A-deficient tumors. Conclusions: ARID1A-deficient cells acquire high sensitivity to PARP inhibition after exposure to exogenously induced DNA breaks such as ionizing radiation. Our findings suggest a novel biologically informed strategy for treating ARID1A-deficient malignancies.

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.

Duplication of mammalian genomes requires replisomes to overcome numerous impediments during passage through open (eu) and condensed (hetero) chromatin. Typically, studies of replication stress characterize mixed populations of challenged and unchallenged replication forks, averaged across S phase, and model a single species of “stressed” replisome. Here, in cells containing potent obstacles to replication, we find two different lesion proximal replisomes. One is bound by the DONSON protein and is more frequent in early S phase, in regions marked by euchromatin. The other interacts with the FANCM DNA translocase, is more prominent in late S phase, and favors heterochromatin. The two forms can also be detected in unstressed cells. ChIP-seq of DNA associated with DONSON or FANCM confirms the bias of the former towards regions that replicate early and the skew of the latter towards regions that replicate late., Eukaryotic replisomes are multiprotein complexes. Here the authors reveal two distinct stressed replisomes, associated with DONSON and FANCM, displaying a bias in replication timing and chromatin domain.

Fanconi Anemia (FA) is characterized by bone marrow failure, congenital abnormalities, and cancer. Of over 20 FA-linked genes, FANCJ uniquely encodes a DNA helicase and mutations are also associated with breast and ovarian cancer. fancj−/− cells are sensitive to DNA interstrand cross-linking (ICL) and replication fork stalling drugs. We delineated the molecular defects of two FA patient-derived FANCJ helicase domain mutations. FANCJ-R707C was compromised in dimerization and helicase processivity, whereas DNA unwinding by FANCJ-H396D was barely detectable. DNA binding and ATP hydrolysis was defective for both FANCJ-R707C and FANCJ-H396D, the latter showing greater reduction. Expression of FANCJ-R707C or FANCJ-H396D in fancj−/− cells failed to rescue cisplatin or mitomycin sensitivity. Live-cell imaging demonstrated a significantly compromised recruitment of FANCJ-R707C to laser-induced DNA damage. However, FANCJ-R707C expressed in fancj-/- cells conferred resistance to the DNA polymerase inhibitor aphidicolin, G-quadruplex ligand telomestatin, or DNA strand-breaker bleomycin, whereas FANCJ-H396D failed. Thus, a minimal threshold of FANCJ catalytic activity is required to overcome replication stress induced by aphidicolin or telomestatin, or to repair bleomycin-induced DNA breakage. These findings have implications for therapeutic strategies relying on DNA cross-link sensitivity or heightened replication stress characteristic of cancer cells.

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

DNA instability disorders are associated with cancer, neurodegeneration, and premature aging. The genome integrity is most vulnerable during S phase during which the replication apparatus encounters many impediments. Among the most severe are interstrand crosslinks (ICLs) which are absolute blocks to helicases, and have always been regarded as absolute blocks to replication. Cells from patients with the genome instability disorder Fanconi Anemia (FA) are highly sensitive to crosslinking agents. We have developed a novel single molecule strategy, based on DNA fibers, to visualize collisions of replication forks with ICLs. Remarkably, we found that DNA synthesis can resume past an ICL, leaving behind still crosslinked parental strands, a process that requires only a few minutes. Traverse of ICLs requires the activity of the Fanconi Anemia translocase FANCM. Furthermore, while the FA “core” complex proteins, which monoubiquitinate the FANCD2 protein, were not required, FANCD2 was. These results indicate that...

Replication of the genome can be slowed or stalled by DNA interstrand crosslinks (ICLs), which are absolute blocks to helix unwinding. Current models propose that fork collisions, either from one (...

Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPis) have shown efficacy in treatment of breast and ovarian cancers with hereditary deletions of BRCA1/2, but the high promise of these drugs has not yet been realized in sporadic cancers. We present here strong preclinical data for a novel, mechanistically based, combinatorial approach to using DNA methyltransferase inhibitors (DNMTi’s), such as decitabine (DAC) and 5-Azacytidine (5-AZA), with PARP inhibitors (PARPi’s) as a treatment strategy for acute myelogenous leukemias (AML) and estogen-, progesterone- and HER2-receptor negative, or triple negative breast cancer (TNBC). We have previously demonstrated that low doses of 5-AZA and DAC alone show efficacy in AML and TNBC, and propose treatment with PARPi's to enhance sensitivity of cancer cells to DNMTis. The mechanistic rationale for our approach is based upon: 1) data from our group and others showing DNMT1 and PARP1 associate in a complex, and this association increases with DNA damage; 2) the fact that 5-AZA and DAC trap DNMT's led us to hypothesize that these drugs might also increase PARP trapping at DNA damage sites; and 3) the cytotoxicity of the most potent PARPi's (e.g. Talazoparib) appears to correlate with the degree of trapping of PARP1 in chromatin. We find that in cultured human AML and TNBC cells, the DNMTi's (5 to 20 nM DAC or 100 to 200nM 5-AZA) and PARPi's (1 to 10 nM Talazoparib) alone trap PARP into chromatin, and this effect is enhanced when the drugs are combined. In addition, the PARPi-DNMTi combination treatment in TNBC MDA-MB-231 and AML MOLM-14 cell lines resulted in significantly increased DNA double strand breaks (DSBs) and enhanced retention of PARP1 and DNMT1 at laser microirradiation DNA damage sites. Compared with non-tumorigenic MCF10A cells, in TNBC cell lines (N = 4), the combined doses resulted in significant (p Citation Format: Feyruz V. Rassool, Nidal Muvarak, Khadiza Chowdury, Carine Robert, Limin Xia, Eun Yong Choi, Yi Cai, Marina Bellani, Ying Zou, Michael Seidman, Søren Bentzen, Maria Baer, Rena Lapidus, Stephen B. Baylin. Combination of DNA methyltransferase and PARP inhibitors as a novel therapy strategy for poor prognosis acute myeloid leukemia and triple-negative breast cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-205.

SummaryHow homologous chromosomes (homologs) find their partner, pair, and recombine during meiosis constitutes the central phenomenon in eukaryotic genetics. It is widely believed that, in most organisms, SPO11-mediated DNA double-strand breaks (DSBs) introduced during prophase I precede and are required for efficient homolog pairing. We now show that, in the mouse, a significant level of homolog pairing precedes programmed DNA cleavage. Strikingly, this early chromosome pairing still requires SPO11 but is not dependent on its ability to make DSBs or homologous recombination proteins. Intriguingly, SUN1, a protein required for telomere attachment to the nuclear envelope and for post-DSB synapsis, is also required for early pre-DSB homolog pairing. Furthermore, pre-DSB pairing at telomeres persists upon entry into prophase I and is most likely important for initiation of synapsis. Our findings suggest that the DSB-triggered homology search may mainly serve to proofread and stabilize the pre-DSB pairing of homologous chromosomes.