Role of 53BP1 in end protection and DNA synthesis at DNA breaks.

Double-strand break (DSB) repair choice is greatly influenced by the initial processing of DNA ends. 53BP1 limits the formation of recombinogenic single-strand DNA (ssDNA) in BRCA1-deficient cells, leading to defects in homologous recombination (HR). However, the exact mechanisms by which 53BP1 inhibits DSB resection remain unclear. Previous studies have identified two potential pathways: protection against DNA2/EXO1 exonucleases presumably through the Shieldin (SHLD) complex binding to ssDNA, and localized DNA synthesis through the CTC1-STN1-TEN1 (CST) and DNA polymerase α (Polα) to counteract resection. Using a combinatorial approach of END-seq, SAR-seq, and RPA ChIP-seq, we directly assessed the extent of resection, DNA synthesis, and ssDNA, respectively, at restriction enzyme-induced DSBs. We show that, in the presence of 53BP1, Polα-dependent DNA synthesis reduces the fraction of resected DSBs and the resection lengths in G0/G1, supporting a previous model that fill-in synthesis can limit the extent of resection. However, in the absence of 53BP1, Polα activity is sustained on ssDNA yet does not substantially counter resection. In contrast, EXO1 nuclease activity is essential for hyperresection in the absence of 53BP1. Thus, Polα-mediated fill-in partially limits resection in the presence of 53BP1 but cannot counter extensive hyperresection due to the loss of 53BP1 exonuclease blockade. These data provide the first nucleotide mapping of DNA synthesis at resected DSBs and provide insight into the relationship between fill-in polymerases and resection exonucleases.

Human flap endonuclease structures, DNA double-base flipping, and a unified understanding of the FEN1 superfamily.

Flap endonuclease (FEN1), essential for DNA replication and repair, removes RNA and DNA 5' flaps. FEN1 5' nuclease superfamily members acting in nucleotide excision repair (XPG), mismatch repair (EXO1), and homologous recombination (GEN1) paradoxically incise structurally distinct bubbles, ends, or Holliday junctions, respectively. Here, structural and functional analyses of human FEN1:DNA complexes show structure-specific, sequence-independent recognition for nicked dsDNA bent 100° with unpaired 3' and 5' flaps. Above the active site, a helical cap over a gateway formed by two helices enforces ssDNA threading and specificity for free 5' ends. Crystallographic analyses of product and substrate complexes reveal that dsDNA binding and bending, the ssDNA gateway, and double-base unpairing flanking the scissile phosphate control precise flap incision by the two-metal-ion active site. Superfamily conserved motifs bind and open dsDNA; direct the target region into the helical gateway, permitting only nonbase-paired oligonucleotides active site access; and support a unified understanding of superfamily substrate specificity.

EGFR-mediated HSP70 phosphorylation facilitates PCNA association with chromatin and DNA replication.

Efficient DNA replication requires highly coordinated programs for the timely recruitment of protein complexes to DNA replication forks. Defects in this process result in replication stress, which in turn activates cell cycle checkpoints, suppresses cell proliferation and induces apoptosis. In response to persistent cell growth signals that speed up DNA replication processes, cells accelerate the recruitment of DNA replication proteins to avoid DNA replication stress. The mechanisms by which cell growth signals induce processes to facilitate the recruitment of DNA replication proteins onto the replication sites remain unclear. Here, we report that the epidermal growth factor receptor (EGFR) phosphorylates heat shock protein 70 (HSP70) for DNA replication. Such a modification promotes nuclear localization and chromatin association of HSP70, which interacts with the DNA replication coordinator, proliferating cell nuclear antigen (PCNA). HSP70 subsequently facilitates the loading of PCNA onto chromatin. Knockdown or chemical inhibition of HSP70 suppresses PCNA association with chromatin and impairs DNA synthesis and Okazaki fragment maturation, leading to replicative DNA double-strand breaks and apoptosis. Furthermore, chemical inhibition of HSP70 potentiates EGFR-tyrosine kinase inhibitor-induced tumor reduction in vivo. This work expands our understanding of oncogenesis-induced DNA replication processes and provides a foundation for improved treatments for EGFR-mutated lung cancer by simultaneously targeting HSP70.

Structure-specific nucleases: role in Okazaki fragment maturation.

Proper function of structure-specific nucleases is key for faithful Okazaki fragment maturation (OFM) process completion. Deregulation of such nucleases leads to aberrant OFM and causes a spectrum of mutations, some of which may confer survival outcomes under specific stresses and serve as attractive targets for therapeutic intervention in human cancers.

FANCD2 and RAD51 recombinase directly inhibit DNA2 nuclease at stalled replication forks and FANCD2 acts as a novel RAD51 mediator in strand exchange to promote genome stability.

FANCD2 protein, a key coordinator and effector of the interstrand crosslink repair pathway, is also required to prevent excessive nascent strand degradation at hydroxyurea-induced stalled forks. The RAD51 recombinase has also been implicated in regulation of resection at stalled replication forks. The mechanistic contributions of these proteins to fork protection are not well understood. Here, we used purified FANCD2 and RAD51 to study how each protein regulates DNA resection at stalled forks. We characterized three mechanisms of FANCD2-mediated fork protection: (1) The N-terminal domain of FANCD2 inhibits the essential DNA2 nuclease activity by directly binding to DNA2 accounting for over-resection in FANCD2 defective cells. (2) Independent of dimerization with FANCI, FANCD2 itself stabilizes RAD51 filaments to inhibit multiple nucleases, including DNA2, MRE11 and EXO1. (3) Unexpectedly, we uncovered a new FANCD2 function: by stabilizing RAD51 filaments, FANCD2 acts to stimulate the strand exchange activity of RAD51. Our work biochemically explains non-canonical mechanisms by which FANCD2 and RAD51 protect stalled forks. We propose a model in which the strand exchange activity of FANCD2 provides a simple molecular explanation for genetic interactions between FANCD2 and BRCA2 in the FA/BRCA fork protection pathway.

hDNA2 nuclease/helicase promotes centromeric DNA replication and genome stability.

DNA2 is a nuclease/helicase that is involved in Okazaki fragment maturation, replication fork processing, and end resection of DNA double-strand breaks. Similar such helicase activity for resolving secondary structures and structure-specific nuclease activity are needed during DNA replication to process the chromosome-specific higher order repeat units present in the centromeres of human chromosomes. Here, we show that DNA2 binds preferentially to centromeric DNA The nuclease and helicase activities of DNA2 are both essential for resolution of DNA structural obstacles to facilitate DNA replication fork movement. Loss of DNA2-mediated clean-up mechanisms impairs centromeric DNA replication and CENP-A deposition, leading to activation of the ATR DNA damage checkpoints at centromeric DNA regions and late-S/G2 cell cycle arrest. Cells that escape arrest show impaired metaphase plate formation and abnormal chromosomal segregation. Furthermore, the DNA2 inhibitor C5 mimics DNA2 knockout and synergistically kills cancer cells when combined with an ATR inhibitor. These findings provide mechanistic insights into how DNA2 supports replication of centromeric DNA and give further insights into new therapeutic strategies.

Replication fork stability confers chemoresistance in BRCA-deficient cells.

Cells deficient in the Brca1 and Brca2 genes have reduced capacity to repair DNA double-strand breaks by homologous recombination and consequently are hypersensitive to DNA-damaging agents, including cisplatin and poly(ADP-ribose) polymerase (PARP) inhibitors. Here we show that loss of the MLL3/4 complex protein, PTIP, protects Brca1/2-deficient cells from DNA damage and rescues the lethality of Brca2-deficient embryonic stem cells. However, PTIP deficiency does not restore homologous recombination activity at double-strand breaks. Instead, its absence inhibits the recruitment of the MRE11 nuclease to stalled replication forks, which in turn protects nascent DNA strands from extensive degradation. More generally, acquisition of PARP inhibitors and cisplatin resistance is associated with replication fork protection in Brca2-deficient tumour cells that do not develop Brca2 reversion mutations. Disruption of multiple proteins, including PARP1 and CHD4, leads to the same end point of replication fork protection, highlighting the complexities by which tumour cells evade chemotherapeutic interventions and acquire drug resistance.

CtIP maintains stability at common fragile sites and inverted repeats by end resection-independent endonuclease activity.

Chromosomal rearrangements often occur at genomic loci with DNA secondary structures, such as common fragile sites (CFSs) and palindromic repeats. We developed assays in mammalian cells that revealed CFS-derived AT-rich sequences and inverted Alu repeats (Alu-IRs) are mitotic recombination hotspots, requiring the repair functions of carboxy-terminal binding protein (CtBP)-interacting protein (CtIP) and the Mre11/Rad50/Nbs1 complex (MRN). We also identified an endonuclease activity of CtIP that is dispensable for end resection and homologous recombination (HR) at I-SceI-generated "clean" double-strand breaks (DSBs) but is required for repair of DSBs occurring at CFS-derived AT-rich sequences. In addition, CtIP nuclease-defective mutants are impaired in Alu-IRs-induced mitotic recombination. These studies suggest that an end resection-independent CtIP function is important for processing DSB ends with secondary structures to promote HR. Furthermore, our studies uncover an important role of MRN, CtIP, and their associated nuclease activities in protecting CFSs in mammalian cells.

Multiple roles of DNA2 nuclease/helicase in DNA metabolism, genome stability and human diseases.

DNA2 nuclease/helicase is a structure-specific nuclease, 5'-to-3' helicase, and DNA-dependent ATPase. It is involved in multiple DNA metabolic pathways, including Okazaki fragment maturation, replication of 'difficult-to-replicate' DNA regions, end resection, stalled replication fork processing, and mitochondrial genome maintenance. The participation of DNA2 in these different pathways is regulated by its interactions with distinct groups of DNA replication and repair proteins and by post-translational modifications. These regulatory mechanisms induce its recruitment to specific DNA replication or repair complexes, such as DNA replication and end resection machinery, and stimulate its efficient cleavage of various structures, for example, to remove RNA primers or to produce 3' overhangs at telomeres or double-strand breaks. Through these versatile activities at replication forks and DNA damage sites, DNA2 functions as both a tumor suppressor and promoter. In normal cells, it suppresses tumorigenesis by maintaining the genomic integrity. Thus, DNA2 mutations or functional deficiency may lead to cancer initiation. However, DNA2 may also function as a tumor promoter, supporting cancer cell survival by counteracting replication stress. Therefore, it may serve as an ideal target to sensitize advanced DNA2-overexpressing cancers to current chemo- and radiotherapy regimens.

PRMT1-mediated FLT3 arginine methylation promotes maintenance of FLT3-ITD+ acute myeloid leukemia.

The presence of FMS-like receptor tyrosine kinase-3 internal tandem duplication (FLT3-ITD) mutations in patients with acute myeloid leukemia (AML) is associated with poor clinical outcome. FLT3 tyrosine kinase inhibitors (TKIs), although effective in kinase ablation, do not eliminate primitive FLT3-ITD+ leukemia cells, which are potential sources of relapse. Thus, understanding the mechanisms underlying FLT3-ITD+ AML cell persistence is essential to devise future AML therapies. Here, we show that expression of protein arginine methyltransferase 1 (PRMT1), the primary type I arginine methyltransferase, is increased significantly in AML cells relative to normal hematopoietic cells. Genome-wide analysis, coimmunoprecipitation assay, and PRMT1-knockout mouse studies indicate that PRMT1 preferentially cooperates with FLT3-ITD, contributing to AML maintenance. Genetic or pharmacological inhibition of PRMT1 markedly blocked FLT3-ITD+ AML cell maintenance. Mechanistically, PRMT1 catalyzed FLT3-ITD protein methylation at arginine 972/973, and PRMT1 promoted leukemia cell growth in an FLT3 methylation-dependent manner. Moreover, the effects of FLT3-ITD methylation in AML cells were partially due to cross talk with FLT3-ITD phosphorylation at tyrosine 969. Importantly, FLT3 methylation persisted in FLT3-ITD+ AML cells following kinase inhibition, indicating that methylation occurs independently of kinase activity. Finally, in patient-derived xenograft and murine AML models, combined administration of AC220 with a type I PRMT inhibitor (MS023) enhanced elimination of FLT3-ITD+ AML cells relative to AC220 treatment alone. Our study demonstrates that PRMT1-mediated FLT3 methylation promotes AML maintenance and suggests that combining PRMT1 inhibition with FLT3 TKI treatment could be a promising approach to eliminate FLT3-ITD+ AML cells.

RAD51 and mitotic function of mus81 are essential for recovery from low-dose of camptothecin in the absence of the WRN exonuclease.

Stabilization of stalled replication forks prevents excessive fork reversal or degradation, which can undermine genome integrity. The WRN protein is unique among the other human RecQ family members to possess exonuclease activity. However, the biological role of the WRN exonuclease is poorly defined. Recently, the WRN exonuclease has been linked to protection of stalled forks from degradation. Alternative processing of perturbed forks has been associated to chemoresistance of BRCA-deficient cancer cells. Thus, we used WRN exonuclease-deficiency as a model to investigate the fate of perturbed forks undergoing degradation, but in a BRCA wild-type condition. We find that, upon treatment with clinically-relevant nanomolar doses of the Topoisomerase I inhibitor camptothecin, loss of WRN exonuclease stimulates fork inactivation and accumulation of parental gaps, which engages RAD51. Such mechanism affects reinforcement of CHK1 phosphorylation and causes persistence of RAD51 during recovery from treatment. Notably, in WRN exonuclease-deficient cells, persistence of RAD51 correlates with elevated mitotic phosphorylation of MUS81 at Ser87, which is essential to prevent excessive mitotic abnormalities. Altogether, these findings indicate that aberrant fork degradation, in the presence of a wild-type RAD51 axis, stimulates RAD51-mediated post-replicative repair and engagement of the MUS81 complex to limit genome instability and cell death.

TRAF6 mediates human DNA2 polyubiquitination and nuclear localization to maintain nuclear genome integrity.

The multifunctional human DNA2 (hDNA2) nuclease/helicase is required to process DNA ends for homology-directed recombination repair (HDR) and to counteract replication stress. To participate in these processes, hDNA2 must localize to the nucleus and be recruited to the replication or repair sites. However, because hDNA2 lacks the nuclear localization signal that is found in its yeast homolog, it is unclear how its migration into the nucleus is regulated during replication or in response to DNA damage. Here, we report that the E3 ligase TRAF6 binds to and mediates the K63-linked polyubiquitination of hDNA2, increasing the stability of hDNA2 and promoting its nuclear localization. Inhibiting TRAF6-mediated polyubiquitination abolishes the nuclear localization of hDNA2, consequently impairing DNA end resection and HDR. Thus, the current study reveals a mechanism for the regulation of hDNA2 localization and establishes that TRAF6-mediated hDNA2 ubiquitination activates DNA repair pathways to maintain nuclear genome integrity.

ITCH nuclear translocation and H1.2 polyubiquitination negatively regulate the DNA damage response.

The downregulation of the DNA damage response (DDR) enables aggressive tumors to achieve uncontrolled proliferation against replication stress, but the mechanisms underlying this process in tumors are relatively complex. Here, we demonstrate a mechanism through which a distinct E3 ubiquitin ligase, ITCH, modulates DDR machinery in triple-negative breast cancer (TNBC). We found that expression of a nuclear form of ITCH was significantly increased in human TNBC cell lines and tumor specimens. Phosphorylation of ITCH at Ser257 by AKT led to the nuclear localization of ITCH and ubiquitination of H1.2. The ITCH-mediated polyubiquitination of H1.2 suppressed RNF8/RNF168-dependent formation of 53BP1 foci, which plays important roles in DDR. Consistent with these findings, impaired ITCH nuclear translocation and H1.2 polyubiquitination sensitized cells to replication stress and limited cell growth and migration. AKT activation of ITCH-H1.2 axis may confer TNBC cells with a DDR repression to counteract the replication stress and increase cancer cell survivorship and growth potential.

A novel thyroid hormone receptor isoform, TRß2-46, promotes SKP2 expression and retinoblastoma cell proliferation.

Retinoblastoma is a childhood retinal tumor that develops from cone photoreceptor precursors in response to inactivating RB1 mutations and loss of functional RB protein. The cone precursor's response to RB loss involves cell type-specific signaling circuitry that helps to drive tumorigenesis. One component of the cone precursor circuitry, the thyroid hormone receptor ß2 (TRß2), enables the aberrant proliferation of diverse RB-deficient cells in part by opposing the down-regulation of S-phase kinase-associated protein 2 (SKP2) by the more widely expressed and tumor-suppressive TRß1. However, it is unclear how TRß2 opposes TRß1 to enable SKP2 expression and cell proliferation. Here, we show that in human retinoblastoma cells TRß2 mRNA encodes two TRß2 protein isoforms: a predominantly cytoplasmic 54-kDa protein (TRß2-54) corresponding to the well-characterized full-length murine Trß2 and an N-terminally truncated and exclusively cytoplasmic 46-kDa protein (TRß2-46) that starts at Met-79. Whereas TRß2 knockdown decreased SKP2 expression and impaired retinoblastoma cell cycle progression, re-expression of TRß2-46 but not TRß2-54 stabilized SKP2 and restored proliferation to an extent similar to that of ectopic SKP2 restoration. We conclude that TRß2-46 is an oncogenic thyroid hormone receptor isoform that promotes SKP2 expression and SKP2-dependent retinoblastoma cell proliferation.

Sequential posttranslational modifications program FEN1 degradation during cell-cycle progression.

We propose that cell-cycle-dependent timing of FEN1 nuclease activity is essential for cell-cycle progression and the maintenance of genome stability. After DNA replication is complete at the exit point of the S phase, removal of excess FEN1 may be crucial. Here, we report a mechanism that controls the programmed degradation of FEN1 via a sequential cascade of posttranslational modifications. We found that FEN1 phosphorylation stimulated its SUMOylation, which in turn stimulated its ubiquitination and ultimately led to its degradation via the proteasome pathway. Mutations or inhibitors that blocked the modification at any step in this pathway suppressed FEN1 degradation. Critically, the presence of SUMOylation- or ubiquitination-defective, nondegradable FEN1 mutant protein caused accumulation of Cyclin B, delays in the G1 and G2/M phases, and polyploidy. These findings may represent a newly identified regulatory mechanism used by cells to ensure precise cell-cycle progression and to prevent transformation.

Reciprocal regulation of Akt and Oct4 promotes the self-renewal and survival of embryonal carcinoma cells.

Signaling via the Akt serine/threonine protein kinase plays critical roles in the self-renewal of embryonic stem cells and their malignant counterpart, embryonal carcinoma cells (ECCs). Here we show that in ECCs, Akt phosphorylated the master pluripotency factor Oct4 at threonine 235, and that the levels of phosphorylated Oct4 in ECCs correlated with resistance to apoptosis and tumorigenic potential. Phosphorylation of Oct4 increased its stability and facilitated its nuclear localization and its interaction with Sox2, which promoted the transcription of the core stemness genes POU5F1 and NANOG. Furthermore, in ECCs, unphosphorylated Oct4 bound to the AKT1 promoter and repressed its transcription. Phosphorylation of Oct4 by Akt resulted in dissociation of Oct4 from the AKT1 promoter, which activated AKT1 transcription and promoted cell survival. Therefore, a site-specific, posttranslational modification of the Oct4 protein orchestrates the regulation of its stability, subcellular localization, and transcriptional activities, which collectively promotes the survival and tumorigenicity of ECCs.

Structural basis of 5' flap recognition and protein-protein interactions of human flap endonuclease 1.

Human flap endonuclease 1 (hFEN1) is a structure-specific nuclease essential for DNA replication and repair processes. hFEN1 has 5' flap removal activity, as well as gap endonuclease activity that is critical for restarting stalled replication forks. Here, we report the crystal structures of wild-type and mutant hFEN1 proteins in complex with DNA substrates, followed by mutagenesis studies that provide mechanistic insight into the protein-protein interactions of hFEN1. We found that in an α-helix forming the helical gateway of hFEN1 recognizes the 5' flap prior to its threading into the active site for cleavage. We also found that the ß-pin region is rigidified into a short helix in R192F hFEN1-DNA structures, suppressing its gap endonuclease activity and cycle-dependent kinase interactions. Our findings suggest that a single mutation at the primary methylation site can alter the function of hFEN1 and provide insight into the role of the ß-pin region in hFEN1 protein interactions that are essential for DNA replication and repair.

Okazaki fragment maturation involves α-segment error editing by the mammalian FEN1/MutSα functional complex.

During nuclear DNA replication, proofreading-deficient DNA polymerase α (Pol α) initiates Okazaki fragment synthesis with lower fidelity than bulk replication by proofreading-proficient Pol δ or Pol ε. Here, we provide evidence that the exonuclease activity of mammalian flap endonuclease (FEN1) excises Pol α replication errors in a MutSα-dependent, MutLα-independent mismatch repair process we call Pol α-segment error editing (AEE). We show that MSH2 interacts with FEN1 and facilitates its nuclease activity to remove mismatches near the 5' ends of DNA substrates. Mouse cells and mice encoding FEN1 mutations display AEE deficiency, a strong mutator phenotype, enhanced cellular transformation, and increased cancer susceptibility. The results identify a novel role for FEN1 in a specialized mismatch repair pathway and a new cancer etiological mechanism.

Triptolide inhibits Wnt signaling in NSCLC through upregulation of multiple Wnt inhibitory factors via epigenetic modifications to Histone H3.

In the last decade, it has become clear that epigenetic changes act together with genetic mutations to promote virtually every stage of tumorigenesis and cancer progression. This knowledge has triggered searches for "epigenetic drugs" that can be developed into new cancer therapies. Here we report that triptolide reduced lung cancer incidence from 70% to 10% in a Fen1 E160D transgenic mouse model and effectively inhibited cancer growth and metastasis in A549 and H460 mouse xenografts. We found that triptolide induced lung cancer cell apoptosis that was associated with global epigenetic changes to histone 3 (H3). These global epigenetic changes in H3 are correlated with an increase in protein expression of five Wnt inhibitory factors that include WIF1, FRZB, SFRP1, ENY2, and DKK1. Triptolide had no effect on DNA methylation status at any of the CpG islands located in the promoter regions of all five Wnt inhibitory factors. Wnt expression is implicated in promoting the development and progression of many lung cancers. Because of this, the potential to target Wnt signaling with drugs that induce epigenetic modifications provides a new avenue for developing novel therapies for patients with these tumor types.

Role of FEN1 S187 phosphorylation in counteracting oxygen-induced stress and regulating postnatal heart development.

Flap endonuclease 1 (FEN1) phosphorylation is proposed to regulate the action of FEN1 in DNA repair as well as Okazaki fragment maturation. However, the biologic significance of FEN1 phosphorylation in response to DNA damage remains unknown. Here, we report an in vivo role for FEN1 phosphorylation, using a mouse line carrying S187A FEN1, which abolishes FEN1 phosphorylation. Although S187A mouse embryonic fibroblast cells showed normal proliferation under low oxygen levels (2%), the mutant cells accumulated oxidative DNA damage, activated DNA damage checkpoints, and showed G1-phase arrest at atmospheric oxygen levels (21%). This suggests an essential role for FEN1 phosphorylation in repairing oxygen-induced DNA damage and maintaining proper cell cycle progression. Consistently, the mutant cardiomyocytes showed G1-phase arrest due to activation of the p53-mediated DNA damage response at the neonatal stage, which reduces the proliferation potential of the cardiomyocytes and impairs heart development. Nearly 50% of newborns with the S187A mutant died in the first week due to failure to undergo the peroxisome proliferator-activated receptor signaling-dependent switch from glycolysis to fatty acid oxidation. The adult mutant mice developed dilated hearts and showed significantly shorter life spans. Altogether, our results reveal an important role of FEN1 phosphorylation to counteract oxygen-induced stress in the heart during the fetal-to-neonatal transition.-Zhou, L., Dai, H., Wu, J., Zhou, M., Yuan, H., Du, J., Yang, L., Wu, X., Xu, H., Hua, Y., Xu, J., Zheng, L., Shen, B. Role of FEN1 S187 phosphorylation in counteracting oxygen-induced stress and regulating postnatal heart development.