Modulating in vitro lung fibroblast activation via senolysis of senescent human alveolar epithelial cells.

Idiopathic pulmonary fibrosis (IPF) is an age-related disease with poor prognosis and limited therapeutic options. Activation of lung fibroblasts and differentiation to myofibroblasts are the principal effectors of disease pathology, but damage and senescence of alveolar epithelial cells, specifically type II (ATII) cells, has recently been identified as a potential trigger event for the progressive disease cycle. Targeting ATII senescence and the senescence-associated secretory phenotype (SASP) is an attractive therapeutic strategy; however, translatable primary human cell models that enable mechanistic studies and drug development are lacking. Here, we describe a novel system of conditioned medium (CM) transfer from bleomycin-induced senescent primary alveolar epithelial cells (AEC) onto normal human lung fibroblasts (NHLF) that demonstrates an enhanced fibrotic transcriptional and secretory phenotype compared to non-senescent AEC CM treatment or direct bleomycin damage of the NHLFs. In this system, the bleomycin-treated AECs exhibit classical hallmarks of cellular senescence, including SASP and a gene expression profile that resembles aberrant epithelial cells of the IPF lung. Fibroblast activation by CM transfer is attenuated by pre-treatment of senescent AECs with the senolytic Navitoclax and AD80, but not with the standard of care agent Nintedanib or senomorphic JAK-targeting drugs (e.g., ABT-317, ruxolitinib). This model provides a relevant human system for profiling novel senescence-targeting therapeutics for IPF drug development.

Biochemical and functional characterization of the p.A165T missense variant of mitochondrial amidoxime-reducing component 1.

Recent genome-wide association studies have identified a missense variant p.A165T in mitochondrial amidoxime-reducing component 1 (mARC1) that is strongly associated with protection from all-cause cirrhosis and improved prognosis in nonalcoholic steatohepatitis. The precise mechanism of this protective effect is unknown. Substitution of alanine 165 with threonine is predicted to affect mARC1 protein stability and to have deleterious effects on its function. To investigate the mechanism, we have generated a knock-in mutant mARC1 A165T and a catalytically dead mutant C273A (as a control) in human hepatoma HepG2 cells, enabling characterization of protein subcellular distribution, stability, and biochemical functions of the mARC1 mutant protein expressed from its endogenous locus. Compared to WT mARC1, we found that the A165T mutant exhibits significant mislocalization outside of its traditional location anchored in the mitochondrial outer membrane and reduces protein stability, resulting in lower basal levels. We evaluated the involvement of the ubiquitin proteasome system in mARC1 A165T degradation and observed increased ubiquitination and faster degradation of the A165T variant. In addition, we have shown that HepG2 cells carrying the MTARC1 p.A165T variant exhibit lower N-reductive activity on exogenously added amidoxime substrates in vitro. The data from these biochemical and functional assays suggest a mechanism by which the MTARC1 p.A165T variant abrogates enzyme function which may contribute to its protective effect in liver disease.

FANCJ promotes PARP1 activity during DNA replication that is essential in BRCA1 deficient cells.

The effectiveness of poly (ADP-ribose) polymerase inhibitors (PARPi) in creating single-stranded DNA gaps and inducing sensitivity requires the FANCJ DNA helicase. Yet, how FANCJ relates to PARP1 inhibition or trapping, which contribute to PARPi toxicity, remains unclear. Here, we find PARPi effectiveness hinges on S-phase PARP1 activity, which is reduced in FANCJ deficient cells as G-quadruplexes sequester PARP1 and MSH2. Additionally, loss of the FANCJ-MLH1 interaction diminishes PARP1 activity; however, depleting MSH2 reinstates PARPi sensitivity and gaps. Indicating sequestered and trapped PARP1 are distinct, FANCJ loss increases PARPi resistance in cells susceptible to PARP1 trapping. However, with BRCA1 deficiency, the loss of FANCJ mirrors PARP1 loss or inhibition, with the detrimental commonality being loss of S-phase PARP1 activity. These insights underline the crucial role of PARP1 activity during DNA replication in BRCA1 deficient cells and emphasize the importance of understanding drug mechanisms for enhancing therapeutic response.

FANCJ promotes PARP1 activity during DNA replication that is essential in BRCA1 deficient cells.

Single-stranded DNA gaps are postulated to be fundamental to the mechanism of anti-cancer drugs. Gaining insights into their induction could therefore be pivotal for advancing therapeutic strategies. For poly (ADP-ribose) polymerase inhibitors (PARPi) to be effective, the presence of FANCJ helicase is required. However, the relationship between FANCJ dependent gaps and PARP1 catalytic inhibition or trapping-both linked to PARPi toxicity in BRCA deficient cells-is yet to be elucidated. Here, we find that the efficacy of PARPi is contingent on S-phase PARP1 activity, which is compromised in FANCJ deficient cells because PARP1, along with MSH2, is "sequestered" by G-quadruplexes. PARP1's replication activity is also diminished in cells missing a FANCJ-MLH1 interaction, but in such cells, depleting MSH2 can release sequestered PARP1, restoring PARPi-induced gaps and sensitivity. Our observations indicate that sequestered and trapped PARP1 are different chromatin-bound forms, with FANCJ loss increasing PARPi resistance in cells susceptible to canonical PARP1 trapping. However, in BRCA1 null cells, the loss of FANCJ mirrors the effects of PARP1 loss or inhibition, with the common detrimental factor being the loss of PARP1 activity during DNA replication, not trapping. These insights underline the crucial role of PARP1 activity during DNA replication in BRCA deficient cells and emphasize the importance of understanding drug mechanisms for enhancing precision medicine.

Treatment of Sulfur Mustard Corneal Injury by Augmenting the DNA Damage Response (DDR): A Novel Approach.

Sulfur mustard (SM) is a highly reactive organic chemical has been used as a chemical warfare agent and terrorist threat since World War I. The cornea is highly sensitive to SM toxicity and exposure to low vapor doses can cause incapacitating acute injuries. Exposure to higher doses can elicit persistent secondary keratopathies that cause reduced quality of life and impaired or lost vision. Despite a century of research, there are no specific treatments for acute or persistent ocular SM injuries. SM cytotoxicity emerges, in part, through DNA alkylation and double-strand breaks (DSBs). Because DSBs can naturally be repaired by DNA damage response pathways with low efficiency, we hypothesized that enhancing the homologous recombination pathway could pose a novel approach to mitigate SM injury. Here, we demonstrate that a dilithium salt of adenosine diphosphoribose (INV-102) increases protein levels of p53 and Sirtuin 6, upregulates transcription of BRCA1/2, enhances γH2AX focus formation, and promotes assembly of repair complexes at DSBs. Based on in vitro evidence showing INV-102 enhancement of DNA damage response through both p53-dependent and p53-independent pathways, we next tested INV-102 in a rabbit preclinical model of corneal injury. In vivo studies demonstrate a marked reduction in the incidence and severity of secondary keratopathies in INV-102-treated eyes compared with vehicle-treated eyes when treatment was started 24 hours after SM vapor exposure. These results suggest DNA repair mechanisms are a viable therapeutic target for SM injury and suggest topical treatment with INV-102 is a promising approach for SM as well as other conditions associated with DSBs. SIGNIFICANCE STATEMENT: Sulfur mustard gas corneal injury currently has no therapeutic treatment. This study aims to show the therapeutic potential of activating the body's natural DNA damage response to activate tissue repair.

Pgc-1α controls epidermal stem cell fate and skin repair by sustaining NAD+ homeostasis during aging.

OBJECTIVE: The epidermal barrier is renewed by the activation, proliferation, and differentiation of keratinocyte stem cells after injury and aging impedes this repair process through undefined mechanisms. We previously identified a gene signature of metabolic dysfunction in aged murine epidermis, but the precise regulators of epidermal repair and age-related growth defects are not well established. Aged mouse models as well as mice with conditional epidermal loss of the metabolic regulator peroxisome proliferator-activated receptor gamma coactivator-1 alpha (Pgc-1α) were used to explore the cellular pathways which control skin repair after injury and stress. METHODS: Aged mice or those with epidermal Pgc-1α deletion (epiPgc-1α KO) and young or Pgc1afl/fl controls were subjected to wound injury, UVB exposure or the inflammatory agent TPA. In vivo and ex vivo analyses of wound closure, skin structure, cell growth and stem cell differentiation were used to understand changes in epidermal re-growth and repair resulting from aging or Pgc-1α loss. RESULTS: Aging impairs epidermal re-growth during wound healing and results in lower expression of Pgc-1α. Mice with conditional deletion of epidermal Pgc-1α exhibit greater inflammation- and UVB-induced cell differentiation, reduced proliferation, and slower wound healing. epiPgc-1α KO mice also displayed reduced keratinocyte NAD+ levels, shorter telomeres, and greater poly ADP-ribosylation, resulting in enhanced stress-stimulated p53 and p21 signaling. When NAD+ was reduced by Pgc-1α loss or pharmacologic inhibition of NAD+ synthesis, there was reduced stress-induced proliferation, increased differentiation, and protection against DNA damage via enhanced epidermal shedding. Similarly, aged mice exhibit disrupted epidermal NAD+ homeostasis and enhanced p53 activation, resulting in p21 growth arrest after wounding. NAD+ precursor treatment restores epidermal growth from old skin to that of young. CONCLUSIONS: Our studies identify a novel role for epidermal Pgc-1α in controlling epidermal repair via its regulation of cellular NAD+ and downstream effects on p53-driven growth arrest. We also establish that parallel mechanisms are evident in aged epidermis, showing that NAD+ signaling is an important controller of physiologic skin repair and that dysfunction of this pathway contributes to age-related wound repair defects.

Four-Year Disease-Free Remission in a Patient With POLE Mutation-Associated Colorectal Cancer Treated Using Anti-PD-1 Therapy.

The stability of the human genome depends upon a delicate balance between replication by high- and low-fidelity DNA polymerases. Aberrant replication by error-prone polymerases or loss of function of high-fidelity polymerases predisposes to genetic instability and, in turn, cancer. DNA polymerase epsilon (Pol ε) is a high-fidelity, processive polymerase that is responsible for the majority of leading strand synthesis, and mutations in Pol ε have been increasingly associated with various human malignancies. The clinical significance of Pol ε mutations, including how and whether they should influence management decisions, remains poorly understood. In this report, we describe a 24-year-old man with an aggressive stage IV high-grade, poorly differentiated colon carcinoma who experienced a dramatic response to single-agent checkpoint inhibitor immunotherapy after rapidly progressing on standard chemotherapy. His response was complete and durable and has been maintained for more than 48 months. Genetic testing revealed a P286R mutation in the endonuclease domain of POLE and an elevated tumor mutational burden of 126 mutations per megabase, both of which have been previously associated with response to immunotherapy. Interestingly, tumor staining for PD-L1 was negative. This case study highlights the importance of genetic profiling of both early and late-stage cancers, the clinical significance of POLE mutations, and how the interplay between genetic instability and immune-checkpoint blockade can impact clinical decision-making.

Unravelling roles of error-prone DNA polymerases in shaping cancer genomes.

Mutagenesis is a key hallmark and enabling characteristic of cancer cells, yet the diverse underlying mutagenic mechanisms that shape cancer genomes are not understood. This review will consider the emerging challenge of determining how DNA damage response pathways-both tolerance and repair-act upon specific forms of DNA damage to generate mutations characteristic of tumors. DNA polymerases are typically the ultimate mutagenic effectors of DNA repair pathways. Therefore, understanding the contributions of DNA polymerases is critical to develop a more comprehensive picture of mutagenic mechanisms in tumors. Selection of an appropriate DNA polymerase-whether error-free or error-prone-for a particular DNA template is critical to the maintenance of genome stability. We review different modes of DNA polymerase dysregulation including mutation, polymorphism, and over-expression of the polymerases themselves or their associated activators. Based upon recent findings connecting DNA polymerases with specific mechanisms of mutagenesis, we propose that compensation for DNA repair defects by error-prone polymerases may be a general paradigm molding the mutational landscape of cancer cells. Notably, we demonstrate that correlation of error-prone polymerase expression with mutation burden in a subset of patient tumors from The Cancer Genome Atlas can identify mechanistic hypotheses for further testing. We contrast experimental approaches from broad, genome-wide strategies to approaches with a narrower focus on a few hundred base pairs of DNA. In addition, we consider recent developments in computational annotation of patient tumor data to identify patterns of mutagenesis. Finally, we discuss the innovations and future experiments that will develop a more comprehensive portrait of mutagenic mechanisms in human tumors.

Polymerase δ promotes chromosomal rearrangements and imprecise double-strand break repair.

Recent studies have implicated DNA polymerases θ (Pol θ) and ß (Pol ß) as mediators of alternative nonhomologous end-joining (Alt-NHEJ) events, including chromosomal translocations. Here we identify subunits of the replicative DNA polymerase δ (Pol δ) as promoters of Alt-NHEJ that results in more extensive intrachromosomal mutations at a single double-strand break (DSB) and more frequent translocations between two DSBs. Depletion of the Pol δ accessory subunit POLD2 destabilizes the complex, resulting in degradation of both POLD1 and POLD3 in human cells. POLD2 depletion markedly reduces the frequency of translocations with sequence modifications but does not affect the frequency of translocations with exact joins. Using separation-of-function mutants, we show that both the DNA synthesis and exonuclease activities of the POLD1 subunit contribute to translocations. As described in yeast and unlike Pol θ, Pol δ also promotes homology-directed repair. Codepletion of POLD2 with 53BP1 nearly eliminates translocations. POLD1 and POLD2 each colocalize with phosphorylated H2AX at ionizing radiation-induced DSBs but not with 53BP1. Codepletion of POLD2 with either ligase 3 (LIG3) or ligase 4 (LIG4) does not further reduce translocation frequency compared to POLD2 depletion alone. Together, these data support a model in which Pol δ promotes Alt-NHEJ in human cells at DSBs, including translocations.

Multiple Roles for Mono- and Poly(ADP-Ribose) in Regulating Stress Responses.

Although stress-induced synthesis of mono(ADP-ribose) (mADPr) and poly(ADP-ribose) (pADPr) conjugates by pADPr polymerase (PARP) enzymes has been studied extensively, the removal and degradation of pADPr, as well as the fate of ADPr metabolites, have received less attention. The observations that stress-induced pADPr undergoes rapid turnover, and that deficiencies in ADPr degradation phenocopy loss of pADPr synthesis, suggest that ADPr degradation is fundamentally important to the cellular stress response. Recent work has identified several distinct families of pADPr hydrolases that can degrade pADPr to release pADPr or mADPr into the cytoplasm. Further, many stress-response proteins contain ADPr-binding domains that can interact with these metabolites. We discuss how pADPr metabolites generated during pADPr degradation can function as signaling intermediates in processes such as inflammation, apoptosis, and DNA damage responses. These studies highlight that the full cycle of ADPr metabolism, including both synthesis and degradation, is necessary for responses to genotoxic stress.

Parp3 promotes long-range end joining in murine cells.

Chromosomal rearrangements, including translocations, are early and essential events in the formation of many tumors. Previous studies that defined the genetic requirements for rearrangement formation have identified differences between murine and human cells, most notably in the role of classic and alternative nonhomologous end-joining (NHEJ) factors. We reported that poly(ADP)ribose polymerase 3 (PARP3) promotes chromosomal rearrangements induced by endonucleases in multiple human cell types. We show here that in contrast to classic (c-NHEJ) factors, Parp3 also promotes rearrangements in murine cells, including translocations in murine embryonic stem cells (mESCs), class-switch recombination in primary B cells, and inversions in tail fibroblasts that generate Eml4-Alk fusions. In mESCs, Parp3-deficient cells had shorter deletion lengths at translocation junctions. This was corroborated using next-generation sequencing of Eml4-Alk junctions in tail fibroblasts and is consistent with a role for Parp3 in promoting the processing of DNA double-strand breaks. We confirmed a previous report that Parp1 also promotes rearrangement formation. In contrast with Parp3, rearrangement junctions in the absence of Parp1 had longer deletion lengths, suggesting that Parp1 may suppress double-strand break processing. Together, these data indicate that Parp3 and Parp1 promote rearrangements with distinct phenotypes.

Corrigendum: PARP3 is a promoter of chromosomal rearrangements and limits G4 DNA.

This corrects the article DOI: 10.1038/ncomms15110.

PARP3 is a promoter of chromosomal rearrangements and limits G4 DNA.

Chromosomal rearrangements are essential events in the pathogenesis of both malignant and nonmalignant disorders, yet the factors affecting their formation are incompletely understood. Here we develop a zinc-finger nuclease translocation reporter and screen for factors that modulate rearrangements in human cells. We identify UBC9 and RAD50 as suppressors and 53BP1, DDB1 and poly(ADP)ribose polymerase 3 (PARP3) as promoters of chromosomal rearrangements across human cell types. We focus on PARP3 as it is dispensable for murine viability and has druggable catalytic activity. We find that PARP3 regulates G quadruplex (G4) DNA in response to DNA damage, which suppresses repair by nonhomologous end-joining and homologous recombination. Chemical stabilization of G4 DNA in PARP3-/- cells leads to widespread DNA double-strand breaks and synthetic lethality. We propose a model in which PARP3 suppresses G4 DNA and facilitates DNA repair by multiple pathways.

Platinum and PARP Inhibitor Resistance Due to Overexpression of MicroRNA-622 in BRCA1-Mutant Ovarian Cancer.

High-grade serous ovarian carcinomas (HGSOCs) with BRCA1/2 mutations exhibit improved outcome and sensitivity to double-strand DNA break (DSB)-inducing agents (i.e., platinum and poly(ADP-ribose) polymerase inhibitors [PARPis]) due to an underlying defect in homologous recombination (HR). However, resistance to platinum and PARPis represents a significant barrier to the long-term survival of these patients. Although BRCA1/2-reversion mutations are a clinically validated resistance mechanism, they account for less than half of platinum-resistant BRCA1/2-mutated HGSOCs. We uncover a resistance mechanism by which a microRNA, miR-622, induces resistance to PARPis and platinum in BRCA1 mutant HGSOCs by targeting the Ku complex and restoring HR-mediated DSB repair. Physiologically, miR-622 inversely correlates with Ku expression during the cell cycle, suppressing non-homologous end-joining and facilitating HR-mediated DSB repair in S phase. Importantly, high expression of miR-622 in BRCA1-deficient HGSOCs is associated with worse outcome after platinum chemotherapy, indicating microRNA-mediated resistance through HR rescue.

Triplication of a 21q22 region contributes to B cell transformation through HMGN1 overexpression and loss of histone H3 Lys27 trimethylation.

Down syndrome confers a 20-fold increased risk of B cell acute lymphoblastic leukemia (B-ALL), and polysomy 21 is the most frequent somatic aneuploidy among all B-ALLs. Yet the mechanistic links between chromosome 21 triplication and B-ALL remain undefined. Here we show that germline triplication of only 31 genes orthologous to human chromosome 21q22 confers mouse progenitor B cell self renewal in vitro, maturation defects in vivo and B-ALL with either the BCR-ABL fusion protein or CRLF2 with activated JAK2. Chromosome 21q22 triplication suppresses histone H3 Lys27 trimethylation (H3K27me3) in progenitor B cells and B-ALLs, and 'bivalent' genes with both H3K27me3 and H3K4me3 at their promoters in wild-type progenitor B cells are preferentially overexpressed in triplicated cells. Human B-ALLs with polysomy 21 are distinguished by their overexpression of genes marked with H3K27me3 in multiple cell types. Overexpression of HMGN1, a nucleosome remodeling protein encoded on chromosome 21q22 (refs. 3,4,5), suppresses H3K27me3 and promotes both B cell proliferation in vitro and B-ALL in vivo.

BCL2 suppresses PARP1 function and nonapoptotic cell death.

BCL2 suppresses apoptosis by binding the BH3 domain of proapoptotic factors and thereby regulating outer mitochondrial membrane permeabilization. Many tumor types, including B-cell lymphomas and chronic lymphocytic leukemia, are dependent on BCL2 for survival but become resistant to apoptosis after treatment. Here, we identified a direct interaction between the antiapoptotic protein BCL2 and the enzyme PARP1, which suppresses PARP1 enzymatic activity and inhibits PARP1-dependent DNA repair in diffuse large B-cell lymphoma cells. The BH3 mimetic ABT-737 displaced PARP1 from BCL2 in a dose-dependent manner, reestablishing PARP1 activity and DNA repair and promoting nonapoptotic cell death. This form of cell death was unaffected by resistance to single-agent ABT-737 that results from upregulation of antiapoptotic BCL2 family members. On the basis of the ability of BCL2 to suppress PARP1 function, we hypothesized that ectopic BCL2 expression would kill PARP inhibitor-sensitive cells. Strikingly, BCL2 expression reduced the survival of PARP inhibitor-sensitive breast cancer and lung cancer cells by 90% to 100%, and these effects were reversed by ABT-737. Taken together, our findings show that a novel interaction between BCL2 and PARP1 blocks PARP1 enzymatic activity and suppresses PARP1-dependent repair. Targeted disruption of the BCL2-PARP1 interaction therefore may represent a potential therapeutic approach for BCL2-expressing tumors resistant to apoptosis.

c-Jun N-terminal kinase-mediated Rad18 phosphorylation facilitates Polη recruitment to stalled replication forks.

The E3 ubiquitin ligase Rad18 chaperones DNA polymerase η (Polη) to sites of UV-induced DNA damage and monoubiquitinates proliferating cell nuclear antigen (PCNA), facilitating engagement of Polη with stalled replication forks and promoting translesion synthesis (TLS). It is unclear how Rad18 activities are coordinated with other elements of the DNA damage response. We show here that Ser-409 residing in the Polη-binding motif of Rad18 is phosphorylated in a checkpoint kinase 1-dependent manner in genotoxin-treated cells. Recombinant Rad18 was phosphorylated specifically at S409 by c-Jun N-terminal kinase (JNK) in vitro. In UV-treated cells, Rad18 S409 phosphorylation was inhibited by a pharmacological JNK inhibitor. Conversely, ectopic expression of JNK and its upstream kinase mitogen-activated protein kinase kinase 4 led to DNA damage-independent Rad18 S409 phosphorylation. These results identify Rad18 as a novel JNK substrate. A Rad18 mutant harboring a Ser → Ala substitution at S409 was compromised for Polη association and did not redistribute Polη to nuclear foci or promote Polη-PCNA interaction efficiently relative to wild-type Rad18. Rad18 S409A also failed to fully complement the UV sensitivity of Rad18-depleted cells. Taken together, these results show that Rad18 phosphorylation by JNK represents a novel mechanism for promoting TLS and DNA damage tolerance.

Analyzing DNA replication dynamics of genotoxin-treated cells using velocity sedimentation.

Following acquisition of DNA damage S-phase progression may potentially be affected via multiple mechanisms. For example DNA damage-activated signal transduction pathways negatively regulate the initiation of DNA synthesis at unfired origins of replication, a process termed the 'S-phase checkpoint' or the 'intra-S-phase checkpoint'. Additionally, many DNA lesions pose physical barriers to replication forks and therefore inhibit DNA synthesis directly by blocking the elongation of active replicons. Inhibition of DNA synthesis in response to DNA damage is commonly assayed by measuring incorporation of radiolabeled or halogenated nucleotides into bulk genomic DNA. However, these techniques do not distinguish between effects of DNA damage on initiation and elongation phases of DNA synthesis. The velocity sedimentation protocol described here allows investigators to determine the effects of DNA damage on initiation and elongation events. This technique involves labeling replicating DNA with (3)H-thymidine, then analyzing the size distribution of labeled ssDNAs based on their differential density sedimentation profiles after centrifugation through alkaline sucrose gradients. Determining the relative abundance and growth rates of small nascent ssDNAs provides an index of initiation and elongation events, respectively. Therefore, analysis of replication dynamics using velocity sedimentation provides a potentially valuable tool for assaying S-phase checkpoints as well as other aspects of DNA replication.

Phosphorylated Rad18 directs DNA polymerase η to sites of stalled replication.

The E3 ubiquitin ligase Rad18 guides DNA Polymerase eta (Polη) to sites of replication fork stalling and mono-ubiquitinates proliferating cell nuclear antigen (PCNA) to facilitate binding of Y family trans-lesion synthesis (TLS) DNA polymerases during TLS. However, it is unclear exactly how Rad18 is regulated in response to DNA damage and how Rad18 activity is coordinated with progression through different phases of the cell cycle. Here we identify Rad18 as a novel substrate of the essential protein kinase Cdc7 (also termed Dbf4/Drf1-dependent Cdc7 kinase [DDK]). A serine cluster in the Polη-binding motif of Rad 18 is phosphorylated by DDK. Efficient association of Rad18 with Polη is dependent on DDK and is necessary for redistribution of Polη to sites of replication fork stalling. This is the first demonstration of Rad18 regulation by direct phosphorylation and provides a novel mechanism for integration of S phase progression with postreplication DNA repair to maintain genome stability.

A novel role for Fanconi anemia (FA) pathway effector protein FANCD2 in cell cycle progression of untransformed primary human cells.

Fanconi Anemia (FA) is a cancer-susceptibility syndrome characterized by cellular sensitivity to DNA inter-strand cross-link (ICL)-inducing agents. The Fanconia Anemia D2 (FANCD2) protein is implicated in repair of various forms of DNA damage including ICLs. Studies with replicating extracts from Xenopus eggs indicate a role for FANCD2 in processing and repair of DNA replication-associated double stranded breaks (DSB). We have investigated the role of FANCD2 in cell cycle progression of cultured human cells. Similar to Xenopus cell-free extracts, we show that chromatin association of FANCD2 in human cells is coupled to ongoing DNA replication. siRNA depletion experiments demonstrate that FANCD2 is necessary for efficient DNA synthesis. However, in contrast with Xenopus extracts, FANCD2-deficiency does not elicit a DNA damage response, and does not affect the elongation phase of DNA synthesis, suggesting that FANCD2 is dispensable for repair of replication-associated DNA damage. Using synchronized cultures of primary untransformed human dermal fibroblasts we demonstrate that FANCD2 is necessary for efficient initiation of DNA synthesis. Taken together, our results suggest a novel role for the FA pathway in regulation of DNA synthesis and cell cycle progression. Inefficient DNA replication may contribute to the genome instability and cancer-propensity of FA patients.