Purification and biochemical characterization of the G4 resolvase and DNA helicase FANCJ.

G-quadruplex (G4) DNA or RNA poses a unique nucleic acid structure in genomic transactions. Because of the unique topology presented by G4, cells have exquisite mechanisms and pathways to metabolize G4 that arise in guanine-rich regions of the genome such as telomeres, promoter regions, ribosomal DNA, and other chromosomal elements. G4 resolvases are often represented by a class of molecular motors known as helicases that disrupt the Hoogsteen hydrogen bonds in G4 by harnessing the chemical energy of nucleoside triphosphate hydrolysis. Of special interest to researchers in the field, including us, is the human FANCJ DNA helicase that efficiently resolves G4 DNA structures. Notably, FANCJ mutations are linked to Fanconi Anemia and are prominent in breast and ovarian cancer. Since our discovery that FANCJ efficiently resolves G4 DNA structures 15 years ago, we and other labs have characterized mechanistic aspects of FANCJ-catalyzed G4 resolution and its biological importance in genomic integrity and cellular DNA replication. In addition to its G4 resolvase function, FANCJ is also a classic DNA helicase that acts on conventional duplex DNA structures, which are relevant to the enzyme's role in interstrand cross link repair, double-strand break repair via homologous recombination, and response to replication stress. Here, we describe detailed procedures for the purification of recombinant FANCJ protein and characterization of its G4 resolvase and duplex DNA helicase activity.

PHOSPHORYLATION-DEPENDENT ASSOCIATION OF WRN WITH RPA IS REQUIRED FOR RECOVERY OF REPLICATION FORKS STALLED AT SECONDARY DNA STRUCTURES.

The WRN protein mutated in the hereditary premature aging disorder Werner syndrome plays a vital role in handling, processing, and restoring perturbed replication forks. One of its most abundant partners, Replication Protein A (RPA), has been shown to robustly enhance WRN helicase activity in specific cases when tested in vitro. However, the significance of RPA-binding to WRN at replication forks in vivo has remained largely unexplored. In this study, we have identified several conserved phosphorylation sites in the acidic domain of WRN that are targeted by Casein Kinase 2 (CK2). Surprisingly, these phosphorylation sites are essential for the interaction between WRN and RPA, both in vitro and in human cells. By characterizing a CK2-unphosphorylatable WRN mutant that lacks the ability to bind RPA, we have determined that the WRN-RPA complex plays a critical role in fork recovery after replication stress whereas the WRN-RPA interaction is not necessary for the processing of replication forks or preventing DNA damage when forks stall or collapse. When WRN fails to bind RPA, fork recovery is impaired, leading to the accumulation of single-stranded DNA gaps in the parental strands, which are further enlarged by the structure-specific nuclease MRE11. Notably, RPA-binding by WRN and its helicase activity are crucial for countering the persistence of G4 structures after fork stalling. Therefore, our findings reveal for the first time a novel role for the WRN-RPA interaction to facilitate fork restart, thereby minimizing G4 accumulation at single-stranded DNA gaps and suppressing accumulation of unreplicated regions that may lead to MUS81-dependent double-strand breaks requiring efficient repair by RAD51 to prevent excessive DNA damage.

Werner syndrome protein works as a dimer for unwinding and replication fork regression.

The determination of the oligomeric state of functional enzymes is essential for the mechanistic understanding of their catalytic activities. RecQ helicases have diverse biochemical activities, but it is still unclear how their activities are related to their oligomeric states. We use single-molecule multi-color fluorescence imaging to determine the oligomeric states of Werner syndrome protein (WRN) during its unwinding and replication fork regression activities. We reveal that WRN binds to a forked DNA as a dimer, and unwinds it without any change of its oligomeric state. In contrast, WRN binds to a replication fork as a tetramer, and is dimerized during activation of replication fork regression. By selectively inhibiting the helicase activity of WRN on specific strands, we reveal how the active dimers of WRN distinctly use the energy of ATP hydrolysis for repetitive unwinding and replication fork regression.

CDK2 phosphorylation of Werner protein (WRN) contributes to WRN's DNA double-strand break repair pathway choice.

Werner syndrome (WS) is an accelerated aging disorder characterized by genomic instability, which is caused by WRN protein deficiency. WRN participates in DNA metabolism including DNA repair. In a previous report, we showed that WRN protein is recruited to laser-induced DNA double-strand break (DSB) sites during various stages of the cell cycle with similar intensities, supporting that WRN participates in both non-homologous end joining (NHEJ) and homologous recombination (HR). Here, we demonstrate that the phosphorylation of WRN by CDK2 on serine residue 426 is critical for WRN to make its DSB repair pathway choice between NHEJ and HR. Cells expressing WRN engineered to mimic the unphosphorylated or phosphorylation state at serine 426 showed abnormal DSB recruitment, altered RPA interaction, strand annealing, and DSB repair activities. The CDK2 phosphorylation on serine 426 stabilizes WRN's affinity for RPA, likely increasing its long-range resection at the end of DNA strands, which is a crucial step for HR. Collectively, the data shown here demonstrate that a CDK2-dependent phosphorylation of WRN regulates DSB repair pathway choice and cell cycle participation.

LEO1 is a partner for Cockayne syndrome protein B (CSB) in response to transcription-blocking DNA damage.

Cockayne syndrome (CS) is an autosomal recessive genetic disorder characterized by photosensitivity, developmental defects, neurological abnormalities, and premature aging. Mutations in CSA (ERCC8), CSB (ERCC6), XPB, XPD, XPG, XPF (ERCC4) and ERCC1 can give rise to clinical phenotypes resembling classic CS. Using a yeast two-hybrid (Y2H) screening approach, we identified LEO1 (Phe381-Ser568 region) as an interacting protein partner of full-length and C-terminal (Pro1010-Cys1493) CSB in two independent screens. LEO1 is a member of the RNA polymerase associated factor 1 complex (PAF1C) with roles in transcription elongation and chromatin modification. Supportive of the Y2H results, purified, recombinant LEO1 and CSB directly interact in vitro, and the two proteins exist in a common complex within human cells. In addition, fluorescently tagged LEO1 and CSB are both recruited to localized DNA damage sites in human cells. Cell fractionation experiments revealed a transcription-dependent, coordinated association of LEO1 and CSB to chromatin following either UVC irradiation or cisplatin treatment of HEK293T cells, whereas the response to menadione was distinct, suggesting that this collaboration occurs mainly in the context of bulky transcription-blocking lesions. Consistent with a coordinated interaction in DNA repair, LEO1 knockdown or knockout resulted in reduced CSB recruitment to chromatin, increased sensitivity to UVC light and cisplatin damage, and reduced RNA synthesis recovery and slower excision of cyclobutane pyrimidine dimers following UVC irradiation; the absence of CSB resulted in diminished LEO1 recruitment. Our data indicate a reciprocal communication between CSB and LEO1 in the context of transcription-associated DNA repair and RNA transcription recovery.

DNA polymerase ß outperforms DNA polymerase γ in key mitochondrial base excision repair activities.

DNA polymerase beta (POLß), well known for its role in nuclear DNA base excision repair (BER), has been shown to be present in the mitochondria of several different cell types. Here we present a side-by-side comparison of BER activities of POLß and POLγ, the mitochondrial replicative polymerase, previously thought to be the only mitochondrial polymerase. We find that POLß is significantly more proficient at single-nucleotide gap filling, both in substrates with ends that require polymerase processing, and those that do not. We also show that POLß has a helicase-independent functional interaction with the mitochondrial helicase, TWINKLE. This interaction stimulates strand-displacement synthesis, but not single-nucleotide gap filling. Importantly, we find that purified mitochondrial extracts from cells lacking POLß are severely deficient in processing BER intermediates, suggesting that mitochondrially localized DNA POLß may be critical for cells with high energetic demands that produce greater levels of oxidative stress and therefore depend upon efficient BER for mitochondrial health.

A high-throughput screen to identify novel small molecule inhibitors of the Werner Syndrome Helicase-Nuclease (WRN).

Werner syndrome (WS), an autosomal recessive genetic disorder, displays accelerated clinical symptoms of aging leading to a mean lifespan less than 50 years. The WS helicase-nuclease (WRN) is involved in many important pathways including DNA replication, recombination and repair. Replicating cells are dependent on helicase activity, leading to the pursuit of human helicases as potential therapeutic targets for cancer treatment. Small molecule inhibitors of DNA helicases can be used to induce synthetic lethality, which attempts to target helicase-dependent compensatory DNA repair pathways in tumor cells that are already genetically deficient in a specific pathway of DNA repair. Alternatively, helicase inhibitors may be useful as tools to study the specialized roles of helicases in replication and DNA repair. In this study, approximately 350,000 small molecules were screened based on their ability to inhibit duplex DNA unwinding by a catalytically active WRN helicase domain fragment in a high-throughput fluorometric assay to discover new non-covalent small molecule inhibitors of the WRN helicase. Select compounds were screened to exclude ones that inhibited DNA unwinding by other helicases in the screen, bound non-specifically to DNA, acted as irreversible inhibitors, or possessed unfavorable chemical properties. Several compounds were tested for their ability to impair proliferation of cultured tumor cells. We observed that two of the newly identified WRN helicase inhibitors inhibited proliferation of cancer cells in a lineage-dependent manner. These studies represent the first high-throughput screen for WRN helicase inhibitors and the results have implications for anti-cancer strategies targeting WRN in different cancer cells and genetic backgrounds.

Multiple RPAs make WRN syndrome protein a superhelicase.

RPA is known to stimulate the helicase activity of Werner syndrome protein (WRN), but the exact stimulation mechanism is not understood. We use single-molecule FRET and magnetic tweezers to investigate the helicase activity of WRN and its stimulation by RPA. We show that WRN alone is a weak helicase which repetitively unwind just a few tens of base pairs, but that binding of multiple RPAs to the enzyme converts WRN into a superhelicase that unidirectionally unwinds double-stranded DNA more than 1 kb. Our study provides a good case in which the activity and biological functions of the enzyme may be fundamentally altered by the binding of cofactors.

Natural polyphenols as sirtuin 6 modulators.

Flavonoids are polyphenolic secondary metabolites synthesized by plants and fungus with various pharmacological effects. Due to their plethora of biological activities, they have been studied extensively in drug development. They have been shown to modulate the activity of a NAD+-dependent histone deacetylase, SIRT6. Because SIRT6 has been implicated in longevity, metabolism, DNA-repair, and inflammatory response reduction, it is an interesting target in inflammatory and metabolic diseases as well as in cancer. Here we show, that flavonoids can alter SIRT6 activity in a structure dependent manner. Catechin derivatives with galloyl moiety displayed significant inhibition potency against SIRT6 at 10 µM concentration. The most potent SIRT6 activator, cyanidin, belonged to anthocyanidins, and produced a 55-fold increase in SIRT6 activity compared to the 3-10 fold increase for the others. Cyanidin also significantly increased SIRT6 expression in Caco-2 cells. Results from the docking studies indicated possible binding sites for the inhibitors and activators. Inhibitors likely bind in a manner that could disturb NAD+ binding. The putative activator binding site was found next to a loop near the acetylated peptide substrate binding site. In some cases, the activators changed the conformation of this loop suggesting that it may play a role in SIRT6 activation.

Cell cycle-dependent phosphorylation regulates RECQL4 pathway choice and ubiquitination in DNA double-strand break repair.

Pathway choice within DNA double-strand break (DSB) repair is a tightly regulated process to maintain genome integrity. RECQL4, deficient in Rothmund-Thomson Syndrome, promotes the two major DSB repair pathways, non-homologous end joining (NHEJ) and homologous recombination (HR). Here we report that RECQL4 promotes and coordinates NHEJ and HR in different cell cycle phases. RECQL4 interacts with Ku70 to promote NHEJ in G1 when overall cyclin-dependent kinase (CDK) activity is low. During S/G2 phases, CDK1 and CDK2 (CDK1/2) phosphorylate RECQL4 on serines 89 and 251, enhancing MRE11/RECQL4 interaction and RECQL4 recruitment to DSBs. After phosphorylation, RECQL4 is ubiquitinated by the DDB1-CUL4A E3 ubiquitin ligase, which facilitates its accumulation at DSBs. Phosphorylation of RECQL4 stimulates its helicase activity, promotes DNA end resection, increases HR and cell survival after ionizing radiation, and prevents cellular senescence. Collectively, we propose that RECQL4 modulates the pathway choice of NHEJ and HR in a cell cycle-dependent manner.

The Identification of a SIRT6 Activator from Brown Algae Fucus distichus.

Brown seaweeds contain many bioactive compounds, including polyphenols, polysaccharides, fucosterol, and fucoxantin. These compounds have several biological activities, including anti-inflammatory, hepatoprotective, anti-tumor, anti-hypertensive, and anti-diabetic activity, although in most cases their mechanisms of action are not understood. In this study, extracts generated from five brown algae (Fucus dichitus, Fucus vesiculosus (Linnaeus), Cytoseira tamariscofolia, Cytoseira nodacaulis, Alaria esculenta) were tested for their ability to activate SIRT6 resulting in H3K9 deacetylation. Three of the five macroalgal extracts caused a significant increase of H3K9 deacetylation, and the effect was most pronounced for F. dichitus. The compound responsible for this in vitro activity was identified by mass spectrometry as fucoidan.

Active Control of Repetitive Structural Transitions between Replication Forks and Holliday Junctions by Werner Syndrome Helicase.

The reactivation of stalled DNA replication via fork regression invokes Holliday junction formation, branch migration, and the recovery of the replication fork after DNA repair or error-free DNA synthesis. The coordination mechanism for these DNA structural transitions by molecular motors, however, remains unclear. Here we perform single-molecule fluorescence experiments with Werner syndrome protein (WRN) and model replication forks. The Holliday junction is readily formed once the lagging arm is unwound, and migrated unidirectionally with 3.2 ± 0.03 bases/s velocity. The recovery of the replication fork was controlled by branch migration reversal of WRN, resulting in repetitive fork regression. The Holliday junction formation, branch migration, and migration direction reversal are all ATP dependent, revealing that WRN uses the energy of ATP hydrolysis to actively coordinate the structural transitions of DNA.

SLX4 contributes to telomere preservation and regulated processing of telomeric joint molecule intermediates.

SLX4 assembles a toolkit of endonucleases SLX1, MUS81 and XPF, which is recruited to telomeres via direct interaction of SLX4 with TRF2. Telomeres present an inherent obstacle for DNA replication and repair due to their high propensity to form branched DNA intermediates. Here we provide novel insight into the mechanism and regulation of the SLX4 complex in telomere preservation. SLX4 associates with telomeres throughout the cell cycle, peaking in late S phase and under genotoxic stress. Disruption of SLX4's interaction with TRF2 or SLX1 and SLX1's nuclease activity independently causes telomere fragility, suggesting a requirement of the SLX4 complex for nucleolytic resolution of branched intermediates during telomere replication. Indeed, the SLX1-SLX4 complex processes a variety of telomeric joint molecules in vitro. The nucleolytic activity of SLX1-SLX4 is negatively regulated by telomeric DNA-binding proteins TRF1 and TRF2 and is suppressed by the RecQ helicase BLM in vitro. In vivo, in the presence of functional BLM, telomeric circle formation and telomere sister chromatid exchange, both arising out of nucleolytic processing of telomeric homologous recombination intermediates, are suppressed. We propose that the SLX4-toolkit is a telomere accessory complex that, in conjunction with other telomere maintenance proteins, ensures unhindered, but regulated telomere maintenance.

Regulation of the human Suv3 helicase on DNA by inorganic cofactors.

Mitochondria are essential organelles and consequently proper expression and maintenance of the mitochondrial genome are indispensable for proper cell function. The mitochondrial Suv3 (SUPV3L1) helicase is known to have a central role in mitochondrial RNA metabolism and to be essential for maintenance of mitochondrial DNA stability. Here we have performed biochemical investigations to determine the potential regulation of the human Suv3 (hSuv3) helicase function by inorganic cofactors. We find that hSuv3 helicase and ATPase activity in vitro is strictly dependent on the presence of specific divalent cations. Interestingly, we show that divalent cations and nucleotide concentration have a direct effect on helicase substrate stability. Also, hSuv3 helicase is able to utilize several different nucleotide cofactors including both NTPs and dNTPs. Intriguingly, the potency of the individual nucleotide as energy source for hSuv3 unwinding differed depending on the included divalent cation and nucleotide concentration. At low concentrations, all four NTPs could support helicase activity with varying effectiveness depending on the included divalent cation. However, at higher nucleotide concentrations, only ATP was able to elicit the helicase activity of hSuv3. Consequently, we speculate that the capacity of hSuv3 DNA unwinding activity might be sensitive to the local availability of specific inorganic cofactors.

Functional deficit associated with a missense Werner syndrome mutation.

Werner syndrome (WS) is a rare autosomal recessive disorder caused by mutations in the WRN gene. WRN helicase, a member of the RecQ helicase family, is involved in various DNA metabolic pathways including DNA replication, recombination, DNA repair and telomere maintenance. In this study, we have characterized the G574R missense mutation, which was recently identified in a WS patient. Our biochemical experiments with purified mutant recombinant WRN protein showed that the G574R mutation inhibits ATP binding, and thereby leads to significant decrease in helicase activity. Exonuclease activity of the mutant protein was not significantly affected, whereas its single strand DNA annealing activity was higher than that of wild type. Deficiency in the helicase activity of the mutant may cause defects in replication and other DNA metabolic processes, which in turn could be responsible for the Werner syndrome phenotype in the patient. In contrast to the usual appearance of WS, the G574R patient has normal stature. Thus the short stature normally associated with WS may not be due to helicase deficiency.

The helicase and ATPase activities of RECQL4 are compromised by mutations reported in three human patients.

RECQL4 is one of five members of the human RecQ helicase family, and is implicated in three syndromes displaying accelerating aging, developmental abnormalities and a predisposition to cancer. In this study, we purified three variants of RECQL4 carrying previously reported patient mutations. These three mutant proteins were analyzed for the known biochemical activities of RECQL4: DNA binding, unwinding of duplex DNA, ATP hydrolysis and annealing of simplex DNA. Further, the mutant proteins were evaluated for stability and recruitment to sites of laser-induced DNA damage. One mutant was helicase-dead, had marginal ATPase activity and may be structurally compromised, while the other two showed greatly reduced helicase and ATPase activities. The remaining biochemical activities and ability to recruit to damage sites were not significantly impaired for any of the mutants. Our findings demonstrate a consistent pattern of functional deficiency and provide further support for a helicase-dependent cellular function of RECQL4 in addition to its N-terminus-dependent role in initiation of replication, a function that may underlie the phenotype of RECQL4-linked disease.

RAPADILINO RECQL4 mutant protein lacks helicase and ATPase activity.

The RecQ family of helicases has been shown to play an important role in maintaining genomic stability. In humans, this family has five members and mutations in three of these helicases, BLM, WRN and RECQL4, are associated with disease. Alterations in RECQL4 are associated with three diseases, Rothmund-Thomson syndrome, Baller-Gerold syndrome, and RAPADILINO syndrome. One of the more common mutations found in RECQL4 is the RAPADILINO mutation, c.1390+2delT which is a splice-site mutation leading to an in-frame skipping of exon 7 resulting in 44 amino acids being deleted from the protein (p.Ala420-Ala463del). In order to characterize the RAPADILINO RECQL4 mutant protein, it was expressed in bacteria and purified using an established protocol. Strand annealing, helicase, and ATPase assays were conducted to characterize the protein's activities relative to WT RECQL4. Here we show that strand annealing activity in the absence of ATP is unchanged from that of WT RECQL4. However, the RAPADILINO protein variant lacks helicase and ssDNA-stimulated ATPase activity. These observations help explain the underlying molecular etiology of the disease and our findings provide insight into the genotype and phenotype association among RECQL4 syndromes.

DNA binding residues in the RQC domain of Werner protein are critical for its catalytic activities.

Werner protein (WRN), member of the RecQ helicase family, is a helicase and exonuclease, and participates in multiple DNA metabolic processes including DNA replication, recombination and DNA repair. Mutations in the WRN gene cause Werner syndrome, associated with premature aging, genome instability and cancer predisposition. The RecQ C-terminal (RQC) domain of WRN, containing α2-α3 loop and ß-wing motifs, is important for DNA binding and for many protein interactions. To better understand the critical functions of this domain, we generated recombinant WRN proteins (using a novel purification scheme) with mutations in Arg-993 within the α2-α3 loop of the RQC domain and in Phe-1037 of the -wing motif. We then studied the catalytic activities and DNA binding of these mutant proteins as well as some important functional protein interactions. The mutant proteins were defective in DNA binding and helicase activity, and interestingly, they had deficient exonuclease activity and strand annealing function. The RQC domain of WRN has not previously been implicated in exonuclease or annealing activities. The mutant proteins could not stimulate NEIL1 incision activity as did the wild type. Thus, the Arg-993 and Phe-1037 in the RQC domain play essential roles in catalytic activity, and in functional interactions mediated by WRN.

The human RecQ helicases BLM and RECQL4 cooperate to preserve genome stability.

Bacteria and yeast possess one RecQ helicase homolog whereas humans contain five RecQ helicases, all of which are important in preserving genome stability. Three of these, BLM, WRN and RECQL4, are mutated in human diseases manifesting in premature aging and cancer. We are interested in determining to which extent these RecQ helicases function cooperatively. Here, we report a novel physical and functional interaction between BLM and RECQL4. Both BLM and RECQL4 interact in vivo and in vitro. We have mapped the BLM interacting site to the N-terminus of RECQL4, comprising amino acids 361-478, and the region of BLM encompassing amino acids 1-902 interacts with RECQL4. RECQL4 specifically stimulates BLM helicase activity on DNA fork substrates in vitro. The in vivo interaction between RECQL4 and BLM is enhanced during the S-phase of the cell cycle, and after treatment with ionizing radiation. The retention of RECQL4 at DNA double-strand breaks is shortened in BLM-deficient cells. Further, depletion of RECQL4 in BLM-deficient cells leads to reduced proliferative capacity and an increased frequency of sister chromatid exchanges. Together, our results suggest that BLM and RECQL4 have coordinated activities that promote genome stability.

RECQL5 cooperates with Topoisomerase II alpha in DNA decatenation and cell cycle progression.

DNA decatenation mediated by Topoisomerase II is required to separate the interlinked sister chromatids post-replication. SGS1, a yeast homolog of the human RecQ family of helicases interacts with Topoisomerase II and plays a role in chromosome segregation, but this functional interaction has yet to be identified in higher organisms. Here, we report a physical and functional interaction of Topoisomerase IIα with RECQL5, one of five mammalian RecQ helicases, during DNA replication. Direct interaction of RECQL5 with Topoisomerase IIα stimulates the decatenation activity of Topoisomerase IIα. Consistent with these observations, RECQL5 co-localizes with Topoisomerase IIα during S-phase of the cell cycle. Moreover, cells with stable depletions of RECQL5 display a slow proliferation rate, a G2/M cell cycle arrest and late S-phase cycling defects. Metaphase spreads generated from RECQL5-depleted cells exhibit undercondensed and entangled chromosomes. Further, RECQL5-depleted cells activate a G2/M checkpoint and undergo apoptosis. These phenotypes are similar to those observed when Topoisomerase II catalytic activity is inhibited. These results reveal an important role for RECQL5 in the maintenance of genomic stability and a new insight into the decatenation process.