The Response of the Replication Apparatus to Leading Template Strand Blocks.

Duplication of the genome requires the replication apparatus to overcome a variety of impediments, including covalent DNA adducts, the most challenging of which is on the leading template strand. Replisomes consist of two functional units, a helicase to unwind DNA and polymerases to synthesize it. The helicase is a multi-protein complex that encircles the leading template strand and makes the first contact with a leading strand adduct. The size of the channel in the helicase would appear to preclude transit by large adducts such as DNA: protein complexes (DPC). Here we discuss some of the extensively studied pathways that support replication restart after replisome encounters with leading template strand adducts. We also call attention to recent work that highlights the tolerance of the helicase for adducts ostensibly too large to pass through the central channel.

Imaging the cellular response to an antigen tagged interstrand crosslinking agent.

Immunofluorescence imaging is a standard experimental tool for monitoring the response of cellular factors to DNA damage. Visualizing the recruitment of DNA Damage Response (DDR) components requires high affinity antibodies, which are generally available. In contrast, reagents for the display of the lesions that induce the response are far more limited. Consequently, DDR factor accumulation often serves as a surrogate for damage, without reporting the actual inducing structure. This limitation has practical implications given the importance of the response to DNA reactive drugs such as those used in cancer therapy. These include interstrand crosslink (ICL) forming compounds which are frequently employed clinically. Among them are the psoralens, natural products that form ICLs upon photoactivation and applied therapeutically since antiquity. However, despite multiple attempts, antibodies against psoralen ICLs have not been developed. To overcome this limitation, we developed a psoralen tagged with an antigen for which there are commercial antibodies. In this report we describe our application of the tagged psoralen in imaging experiments, and the unexpected discoveries they revealed.

Visualizing replication fork encounters with DNA interstrand crosslinks.

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.

Replication of the Mammalian Genome by Replisomes Specific for Euchromatin and Heterochromatin.

Replisomes follow a schedule in which replication of DNA in euchromatin is early in S phase while sequences in heterochromatin replicate late. Impediments to DNA replication, referred to as replication stress, can stall replication forks triggering activation of the ATR kinase and downstream pathways. While there is substantial literature on the local consequences of replisome stalling-double strand breaks, reversed forks, or genomic rearrangements-there is limited understanding of the determinants of replisome stalling vs. continued progression. Although many proteins are recruited to stalled replisomes, current models assume a single species of "stressed" replisome, independent of genomic location. Here we describe our approach to visualizing replication fork encounters with the potent block imposed by a DNA interstrand crosslink (ICL) and our discovery of an unexpected pathway of replication restart (traverse) past an intact ICL. Additionally, we found two biochemically distinct replisomes distinguished by activity in different stages of S phase and chromatin environment. Each contains different proteins that contribute to ICL traverse.

Visualization of Replisome Encounters with an Antigen Tagged Blocking Lesion.

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.

FANCJ compensates for RAP80 deficiency and suppresses genomic instability induced by interstrand cross-links.

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.

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

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.

EXD2 Protects Stressed Replication Forks and Is Required for Cell Viability in the Absence of BRCA1/2.

Accurate DNA replication is essential to preserve genomic integrity and prevent chromosomal instability-associated diseases including cancer. Key to this process is the cells' ability to stabilize and restart stalled replication forks. Here, we show that the EXD2 nuclease is essential to this process. EXD2 recruitment to stressed forks suppresses their degradation by restraining excessive fork regression. Accordingly, EXD2 deficiency leads to fork collapse, hypersensitivity to replication inhibitors, and genomic instability. Impeding fork regression by inactivation of SMARCAL1 or removal of RECQ1's inhibition in EXD2-/- cells restores efficient fork restart and genome stability. Moreover, purified EXD2 efficiently processes substrates mimicking regressed forks. Thus, this work identifies a mechanism underpinned by EXD2's nuclease activity, by which cells balance fork regression with fork restoration to maintain genome stability. Interestingly, from a clinical perspective, we discover that EXD2's depletion is synthetic lethal with mutations in BRCA1/2, implying a non-redundant role in replication fork protection.

Loss of ARID1A in Tumor Cells Renders Selective Vulnerability to Combined Ionizing Radiation and PARP Inhibitor Therapy.

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.

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

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.

Imaging cellular responses to antigen tagged DNA damage.

Repair pathways of covalent DNA damage are understood in considerable detail due to decades of brilliant biochemical studies by many investigators. An important feature of these experiments is the defined adduct location on oligonucleotide or plasmid substrates that are incubated with purified proteins or cell free extracts. With some exceptions, this certainty is lost when the inquiry shifts to the response of living mammalian cells to the same adducts in genomic DNA. This reflects the limitation of assays, such as those based on immunofluorescence, that are widely used to follow responding proteins in cells exposed to a DNA reactive compound. The lack of effective reagents for adduct detection means that the proximity between responding proteins and an adduct must be assumed. Since these assumptions can be incorrect, models based on in vitro systems may fail to account for observations made in vivo. Here we discuss the use of a detection tag to address the problem of lesion location, as illustrated by our recent work on replication dependent and independent responses to interstrand crosslinks.

A minimal threshold of FANCJ helicase activity is required for its response to replication stress or double-strand break repair.

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.

Single Molecule Analysis of Laser Localized Psoralen Adducts.

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.

Single Molecule Analysis of Laser Localized Interstrand Crosslinks.

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 (IF), 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 immuno-quantum 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 defined by a UVA laser. The antigen tag can be displayed by conventional IF, as can the recruitment and accumulation of DNA damage response 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.

Integrated transcriptome analysis of mouse spermatogenesis.

BACKGROUND: Differentiation of primordial germ cells into mature spermatozoa proceeds through multiple stages, one of the most important of which is meiosis. Meiotic recombination is in turn a key part of meiosis. To achieve the highly specialized and diverse functions necessary for the successful completion of meiosis and the generation of spermatozoa thousands of genes are coordinately regulated through spermatogenesis. A complete and unbiased characterization of the transcriptome dynamics of spermatogenesis is, however, still lacking. RESULTS: In order to characterize gene expression during spermatogenesis we sequenced eight mRNA samples from testes of juvenile mice from 6 to 38 days post partum. Using gene expression clustering we defined over 1,000 novel meiotically-expressed genes. We also developed a computational de-convolution approach and used it to estimate cell type-specific gene expression in pre-meiotic, meiotic and post-meiotic cells. In addition, we detected 13,000 novel alternative splicing events around 40% of which preserve an open reading frame, and found experimental support for 159 computational gene predictions. A comparison of RNA polymerase II (Pol II) ChIP-Seq signals with RNA-Seq coverage shows that gene expression correlates well with Pol II signals, both at promoters and along the gene body. However, we observe numerous instances of non-canonical promoter usage, as well as intergenic Pol II peaks that potentially delineate unannotated promoters, enhancers or small RNA clusters. CONCLUSIONS: Here we provide a comprehensive analysis of gene expression throughout mouse meiosis and spermatogenesis. Importantly, we find over a thousand of novel meiotic genes and over 5,000 novel potentially coding isoforms. These data should be a valuable resource for future studies of meiosis and spermatogenesis in mammals.

The dual role of HOP2 in mammalian meiotic homologous recombination.

Deletion of Hop2 in mice eliminates homologous chromosome synapsis and disrupts double-strand break (DSB) repair through homologous recombination. HOP2 in vitro shows two distinctive activities: when it is incorporated into a HOP2-MND1 complex it stimulates DMC1 and RAD51 recombination activities and the purified HOP2 alone is proficient in promoting strand invasion. We observed that a fraction of Mnd1(-/-) spermatocytes, which express HOP2 but apparently have inactive DMC1 and RAD51 due to lack of the HOP2-MND1 complex, exhibits a high level of chromosome synapsis and that most DSBs in these spermatocytes are repaired. This suggests that DSB repair catalyzed solely by HOP2 supports homologous chromosome pairing and synapsis. In addition, we show that in vitro HOP2 promotes the co-aggregation of ssDNA with duplex DNA, binds to ssDNA leading to unstacking of the bases, and promotes the formation of a three-strand synaptic intermediate. However, HOP2 shows distinctive mechanistic signatures as a recombinase. Namely, HOP2-mediated strand exchange does not require ATP and, in contrast to DMC1, joint molecules formed by HOP2 are more sensitive to mismatches and are efficiently dissociated by RAD54. We propose that HOP2 may act as a recombinase with specific functions in meiosis.

The DNA translocase FANCM/MHF promotes replication traverse of DNA interstrand crosslinks.

The replicative machinery encounters many impediments, some of which can be overcome by lesion bypass or replication restart pathways, leaving repair for a later time. However, interstrand crosslinks (ICLs), which preclude DNA unwinding, are considered absolute blocks to replication. Current models suggest that fork collisions, either from one or both sides of an ICL, initiate repair processes required for resumption of replication. To test these proposals, we developed a single-molecule technique for visualizing encounters of replication forks with ICLs as they occur in living cells. Surprisingly, the most frequent patterns were consistent with replication traverse of an ICL, without lesion repair. The traverse frequency was strongly reduced by inactivation of the translocase and DNA binding activities of the FANCM/MHF complex. The results indicate that translocase-based mechanisms enable DNA synthesis to continue past ICLs and that these lesions are not always absolute blocks to replication.

Homologous pairing preceding SPO11-mediated double-strand breaks in mice.

How 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.

The expression profile of the major mouse SPO11 isoforms indicates that SPO11beta introduces double strand breaks and suggests that SPO11alpha has an additional role in prophase in both spermatocytes and oocytes.

Both in mice and humans, two major SPO11 isoforms are generated by alternative splicing: SPO11alpha (exon 2 skipped) and SPO11beta. Thus, the alternative splicing event must have emerged before the mouse and human lineages diverged and was maintained during 90 million years of evolution, arguing for an essential role for both isoforms. Here we demonstrate that developmental regulation of alternative splicing at the Spo11 locus governs the sequential expression of SPO11 isoforms in male meiotic prophase. Protein quantification in juvenile mice and in prophase mutants indicates that early spermatocytes synthesize primarily SPO11beta. Estimation of the number of SPO11 dimers (betabeta/alphabeta/alphaalpha) in mutants in which spermatocytes undergo a normal number of double strand breaks but arrest in midprophase due to inefficient repair argues for a role for SPO11beta-containing dimers in introducing the breaks in leptonema. Expression kinetics in males suggested a role for SPO11alpha in pachytene/diplotene spermatocytes. Nevertheless, we found that both alternative transcripts can be detected in oocytes throughout prophase I, arguing against a male-specific function for this isoform. Altogether, our data support a role for SPO11alpha in mid- to late prophase, presumably acting as a topoisomerase, that would be conserved in male and female meiocytes.

SPO11 is required for sex-body formation, and Spo11 heterozygosity rescues the prophase arrest of Atm-/- spermatocytes.

SPO11 introduces double-strand breaks (DSBs) that trigger the phosphorylation of H2AX during meiotic prophase. In mice, SPO11 is strictly required for initiation of meiotic recombination and synapsis, yet SPO11 is still considered to be dispensable for sex-body formation in mouse spermatocytes. We provide conclusive evidence showing that functional SPO11, and consequently recombination and synapsis, are required for phosphorylation of H2AX in the X-Y chromatin and for sex-body formation in mouse spermatocytes. We investigated the role in meiosis of the three kinases [ATM (ataxia telangiectasia mutated), ATR (ataxia-telangiectasia- and Rad-3-related) and DNA-PKcs (DNA-dependent-protein-kinase catalytic subunit)] known to phosphorylate H2AX in mitotic cells. We found that DNA-PKcs can be ruled out as an essential kinase in this process, whereas ATM is strictly required for the chromatin-wide phosphorylation of H2AX occurring in leptotene spermatocytes in response to DSBs. Remarkably, we discovered that Spo11 heterozygosity can rescue the prophase-I-arrest characteristic of ATM-deficient spermatocytes. Characterization of the rescued Atm-/- Spo11+/- mutant indicates that ATM is dispensable for sex-body formation and phosphorylation of H2AX in this subnuclear domain. The co-localization of ATR, phosphorylated H2AX and the sex chromatin observed in the Atm-/- Spo11+/- mutant, along with ATR transcription kinetics during the first wave of spermatogenesis, confirm and expand recent findings indicating that ATR is the kinase involved in H2AX phosphorylation in the sex body.