Sanjay Kumar Bharti

Specialization Among Iron-Sulfur Cluster Helicases to Resolve G-Quadruplex DNA Structures that Threaten Genomic Stability

Background: The Fe-S helicase FANCJ implicated in Fanconi anemia plays important roles in DNA replication and repair. Results: FANCJ, but not the Fe-S XPD or DDX11 helicases, unwinds unimolecular G4 DNA. Conclusion: FANCJ is a specialized Fe-S helicase, preventing G4-induced DNA damage. Significance: FANCJ has a unique role in DNA metabolism to prevent G4 accumulation that causes genomic instability. G-quadruplex (G4) DNA, an alternate structure formed by Hoogsteen hydrogen bonds between guanines in G-rich sequences, threatens genomic stability by perturbing normal DNA transactions including replication, repair, and transcription. A variety of G4 topologies (intra- and intermolecular) can form in vitro, but the molecular architecture and cellular factors influencing G4 landscape in vivo are not clear. Helicases that unwind structured DNA molecules are emerging as an important class of G4-resolving enzymes. The BRCA1-associated FANCJ helicase is among those helicases able to unwind G4 DNA in vitro, and FANCJ mutations are associated with breast cancer and linked to Fanconi anemia. FANCJ belongs to a conserved iron-sulfur (Fe S) cluster family of helicases important for genomic stability including XPD (nucleotide excision repair), DDX11 (sister chromatid cohesion), and RTEL (telomere metabolism), genetically linked to xeroderma pigmentosum/Cockayne syndrome, Warsaw breakage syndrome, and dyskeratosis congenita, respectively. To elucidate the role of FANCJ in genomic stability, its molecular functions in G4 metabolism were examined. FANCJ efficiently unwound in a kinetic and ATPase-dependent manner entropically favored unimolecular G4 DNA, whereas other Fe-S helicases tested did not. The G4-specific ligands Phen-DC3 or Phen-DC6 inhibited FANCJ helicase on unimolecular G4 ∼1000-fold better than bi- or tetramolecular G4 DNA. The G4 ligand telomestatin induced DNA damage in human cells deficient in FANCJ but not DDX11 or XPD. These findings suggest FANCJ is a specialized Fe-S cluster helicase that preserves chromosomal stability by unwinding unimolecular G4 DNA likely to form in transiently unwound single-stranded genomic regions.

Identification and biochemical characterization of a novel mutation in DDX11 causing Warsaw breakage syndrome.

Mutations in the gene encoding the iron–sulfur‐containing DNA helicase DDX11 (ChlR1) were recently identified as a cause of a new recessive cohesinopathy, Warsaw breakage syndrome (WABS), in a single patient with severe microcephaly, pre‐ and postnatal growth retardation, and abnormal skin pigmentation. Here, using homozygosity mapping in a Lebanese consanguineous family followed by exome sequencing, we identified a novel homozygous mutation (c.788G>A [p.R263Q]) in DDX11 in three affected siblings with severe intellectual disability and many of the congenital abnormalities reported in the WABS original case. Cultured lymphocytes from the patients showed increased mitomycin C‐induced chromosomal breakage, as found in WABS. Biochemical studies of purified recombinant DDX11 indicated that the p.R263Q mutation impaired DDX11 helicase activity by perturbing its DNA binding and DNA‐dependent ATP hydrolysis. Our findings thus confirm the involvement of DDX11 in WABS, describe its phenotypical spectrum, and provide novel insight into the structural requirement for DDX11 activity.

DNA sequences proximal to human mitochondrial DNA deletion breakpoints prevalent in human disease form G-quadruplexes, a class of DNA structures inefficiently unwound by the mitochondrial replicative Twinkle helicase.

Background: Mitochondrial DNA deletions are prominent in human genetic disorders and cancer. Results: Predicted mitochondrial G-quadruplex-forming sequences map in close proximity to known deletion breakpoints and form G-quadruplexes in vitro. Conclusion: The mitochondrial replicative helicase Twinkle inefficiently unwinds intra- and intermolecular G-quadruplexes. Significance: Mitochondrial G-quadruplexes are likely to cause genome instability by perturbing replication machinery. Mitochondrial DNA deletions are prominent in human genetic disorders, cancer, and aging. It is thought that stalling of the mitochondrial replication machinery during DNA synthesis is a prominent source of mitochondrial genome instability; however, the precise molecular determinants of defective mitochondrial replication are not well understood. In this work, we performed a computational analysis of the human mitochondrial genome using the “Pattern Finder” G-quadruplex (G4) predictor algorithm to assess whether G4-forming sequences reside in close proximity (within 20 base pairs) to known mitochondrial DNA deletion breakpoints. We then used this information to map G4P sequences with deletions characteristic of representative mitochondrial genetic disorders and also those identified in various cancers and aging. Circular dichroism and UV spectral analysis demonstrated that mitochondrial G-rich sequences near deletion breakpoints prevalent in human disease form G-quadruplex DNA structures. A biochemical analysis of purified recombinant human Twinkle protein (gene product of c10orf2) showed that the mitochondrial replicative helicase inefficiently unwinds well characterized intermolecular and intramolecular G-quadruplex DNA substrates, as well as a unimolecular G4 substrate derived from a mitochondrial sequence that nests a deletion breakpoint described in human renal cell carcinoma. Although G4 has been implicated in the initiation of mitochondrial DNA replication, our current findings suggest that mitochondrial G-quadruplexes are also likely to be a source of instability for the mitochondrial genome by perturbing the normal progression of the mitochondrial replication machinery, including DNA unwinding by Twinkle helicase.

Molecular functions and cellular roles of the ChlR1 (DDX11) helicase defective in the rare cohesinopathy Warsaw breakage syndrome

In 2010, a new recessive cohesinopathy disorder, designated Warsaw breakage syndrome (WABS), was described. The individual with WABS displayed microcephaly, pre- and postnatal growth retardation, and abnormal skin pigmentation. Cytogenetic analysis revealed mitomycin C (MMC)-induced chromosomal breakage; however, an additional sister chromatid cohesion defect was also observed. WABS is genetically linked to bi-allelic mutations in the ChlR1/DDX11 gene which encodes a protein of the conserved family of Iron–Sulfur (Fe–S) cluster DNA helicases. Mutations in the budding yeast ortholog of ChlR1, known as Chl1, were known to cause sister chromatid cohesion defects, indicating a conserved function of the gene. In 2012, three affected siblings were identified with similar symptoms to the original WABS case, and found to have a homozygous mutation in the conserved Fe–S domain of ChlR1, confirming the genetic linkage. Significantly, the clinically relevant mutations perturbed ChlR1 DNA unwinding activity. In addition to its genetic importance in human disease, ChlR1 is implicated in papillomavirus genome maintenance and cancer. Although its precise functions in genome homeostasis are still not well understood, ongoing molecular studies of ChlR1 suggest the helicase plays a critically important role in cellular replication and/or DNA repair.

A Long Noncoding RNA Regulates Sister Chromatid Cohesion

Long noncoding RNAs (lncRNAs) are involved in diverse cellular processes through multiple mechanisms. Here, we describe a previously uncharacterized human lncRNA, CONCR (cohesion regulator noncoding RNA), that is transcriptionally activated by MYC and is upregulated in multiple cancer types. The expression of CONCR is cell cycle regulated, and it is required for cell-cycle progression and DNA replication. Moreover, cells depleted of CONCR show severe defects in sister chromatid cohesion, suggesting an essential role for CONCR in cohesion establishment during cell division. CONCR interacts with and regulates the activity of DDX11, a DNA-dependent ATPase and helicase involved in DNA replication and sister chromatid cohesion. These findings unveil a direct role for an lncRNA in the establishment of sister chromatid cohesion by modulating DDX11 enzymatic activity.

Tim/Timeless, a member of the replication fork protection complex, operates with the Warsaw breakage syndrome DNA helicase DDX11 in the same fork recovery pathway

We present evidence that Tim establishes a physical and functional interaction with DDX11, a super-family 2 iron-sulfur cluster DNA helicase genetically linked to the chromosomal instability disorder Warsaw breakage syndrome. Tim stimulates DDX11 unwinding activity on forked DNA substrates up to 10-fold and on bimolecular anti-parallel G-quadruplex DNA structures and three-stranded D-loop approximately 4–5-fold. Electrophoretic mobility shift assays revealed that Tim enhances DDX11 binding to DNA, suggesting that the observed stimulation derives from an improved ability of DDX11 to interact with the nucleic acid substrate. Surface plasmon resonance measurements indicate that DDX11 directly interacts with Tim. DNA fiber track assays with HeLa cells exposed to hydroxyurea demonstrated that Tim or DDX11 depletion significantly reduced replication fork progression compared to control cells; whereas no additive effect was observed by co-depletion of both proteins. Moreover, Tim and DDX11 are epistatic in promoting efficient resumption of stalled DNA replication forks in hydroxyurea-treated cells. This is consistent with the finding that association of the two endogenous proteins in the cell extract chromatin fraction is considerably increased following hydroxyurea exposure. Overall, our studies provide evidence that Tim and DDX11 physically and functionally interact and act in concert to preserve replication fork progression in perturbed conditions.

Cockayne syndrome group A and B proteins converge on transcription-linked resolution of non-B DNA

Significance In this paper we describe a possible pathogenesis for the accelerated aging disease Cockayne syndrome that entails defective transcription through DNA secondary structures leading to activation of the DNA damage response enzyme poly-ADP-ribose polymerase 1 and downstream mitochondrial derangement. These findings are important because they signify a possible new role of transcription in the resolution of DNA structures that form spontaneously and suggest a possible pathogenesis for this accelerated aging disease. Cockayne syndrome is a neurodegenerative accelerated aging disorder caused by mutations in the CSA or CSB genes. Although the pathogenesis of Cockayne syndrome has remained elusive, recent work implicates mitochondrial dysfunction in the disease progression. Here, we present evidence that loss of CSA or CSB in a neuroblastoma cell line converges on mitochondrial dysfunction caused by defects in ribosomal DNA transcription and activation of the DNA damage sensor poly-ADP ribose polymerase 1 (PARP1). Indeed, inhibition of ribosomal DNA transcription leads to mitochondrial dysfunction in a number of cell lines. Furthermore, machine-learning algorithms predict that diseases with defects in ribosomal DNA (rDNA) transcription have mitochondrial dysfunction, and, accordingly, this is found when factors involved in rDNA transcription are knocked down. Mechanistically, loss of CSA or CSB leads to polymerase stalling at non-B DNA in a neuroblastoma cell line, in particular at G-quadruplex structures, and recombinant CSB can melt G-quadruplex structures. Indeed, stabilization of G-quadruplex structures activates PARP1 and leads to accelerated aging in Caenorhabditis elegans. In conclusion, this work supports a role for impaired ribosomal DNA transcription in Cockayne syndrome and suggests that transcription-coupled resolution of secondary structures may be a mechanism to repress spurious activation of a DNA damage response.

Getting Ready for the Dance: FANCJ Irons Out DNA Wrinkles

Mounting evidence indicates that alternate DNA structures, which deviate from normal double helical DNA, form in vivo and influence cellular processes such as replication and transcription. However, our understanding of how the cellular machinery deals with unusual DNA structures such as G-quadruplexes (G4), triplexes, or hairpins is only beginning to emerge. New advances in the field implicate a direct role of the Fanconi Anemia Group J (FANCJ) helicase, which is linked to a hereditary chromosomal instability disorder and important for cancer suppression, in replication past unusual DNA obstacles. This work sets the stage for significant progress in dissecting the molecular mechanisms whereby replication perturbation by abnormal DNA structures leads to genomic instability. In this review, we focus on FANCJ and its role to enable efficient DNA replication when the fork encounters vastly abundant naturally occurring DNA obstacles, which may have implications for targeting rapidly dividing cancer cells.

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

Abstract 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.

Fine-tuning DNA repair by protein acetylation

Post-translational modifications of target DNA repair proteins may help to regulate the mechanism of DNA damage correction. A good analogy is an automobile tune-up necessary to make a car operate at its optimal capacity (Fig. 1). Although the vehicle may be serviceable, an oil change or spark plug replacement may improve engine performance. In a similar manner, the functionality of the cell’s DNA repair machinery may be boosted by a covalent modification of a target protein responsible for removal of the damaged base to suppress mutation. In a recent work, Piekna-Przybylska et al. assessed biological effects of protein acetylation on DNA repair capacity in human cells. The researchers’ experimental approach was based on flow cytometric analysis of human cells transfected with a reporter DNA plasmid harboring a site-specific oxidative base lesion (8-oxoguanine (8-oxoG)) or base-base mismatch that disrupted coding sequence of a green fluorescent marker gene. The reporter plasmid DNA construct contained both the lesion and a single-strand nick, the latter providing a site for DNA repair machinery (e.g., mismatch repair (MMR)) to load onto the substrate and excise the damaged strand so it can be replaced with normal sequence. The DNA repair assay monitored correction of the abnormal DNA base in cells defective in MMR and/or a major acetyltransferase known as p300. Alternatively, MMR-proficient or deficient cells were pharmacologically modulated with deacetylase or acetyltransferase inhibitors to assess the importance of acetylation status on DNA repair capacity. Pharmacological inhibition of deacetylase activity was observed to up-regulate nick-directed DNA repair of a mismatch in the cell-based assay. Consistent with this finding, cellular exposure to an acetyltransferase inhibitor decreased repair of mismatches or nicks, even in cells genetically defective in MLH1, a key component of the MMR machinery. Presumably, acetylation of a human nuclease involved in replication intermediate processing (e.g., DNA2 implicated in Okazaki fragment maturation) increases repair of duplex DNA containing a nick or mismatch; however, the precise mechanism remains to be determined. Alternatively, acetylation (and other post-translational modifications) of a MMR helicase or DNA polymerase might alter its catalytic DNA efficiency, which in turn affects strand displacement synthesis. Interestingly, pharmacological modulation of acetylation did not affect 8-oxoG correction, suggesting that posttranslational regulatory control of base excision repair (BER), even long patch BER processing of DNA flaps, is distinct from that which operates for mismatch correction. Because p300 acetyltransferase is known to acetylate non-histone and histone proteins, the authors investigated the effect of p300 deficiency on DNA repair and found that cells were significantly compromised in 5-nick directed mismatch correction irrespective of MLH1 mutational status, further suggesting that acetylation promotes excision of the tract containing the mismatched base independent of the classic MMR pathway. However, p300 deficiency exerted no effect on BER of 8-oxoG. This result was unexpected, given a number of in vitro studies showing that BER proteins can be acetylated and that long patch BER in reconstituted cell extract systems was affected by acetylation. The existence of other acetyltransferases engaged in the DNA damage response in human cells (CBP, p300, PCAF, Tip60, MOF) may negate the effect of p300 deficiency on BER, or the scenario may be more complex. It will be important to assess precisely which acetyltransferase(s) serves as the best bull’s eye for DNA repair modulation. A significant advance in the current work was that the scientists addressed the importance of acetylation in DNA repair in living human cells, whereas the majority of previous studies examined the effect of acetylation on DNA replication or repair in vitro using reconstituted systems. Although the plasmid reporter system is valuable in that it focuses on acetylation of DNA repair proteins and likely excludes effects of acetylation on chromatin state and therefore lesion access, it will be important to validate the findings of the current work for clinically relevant chromosomal genomic lesions. This may provide a better understanding of how emerging therapies focused on DNA damage response pathways can be fine-tuned. In addition, lagging strand replication errors could be a suitable target for acetylation-based cancer therapy. If acetylation truly is a direct modulator of DNA repair, it will be relevant at the translational level