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.
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.
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.
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.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
•The fork regression of WRN is readily activated after lagging arm unwinding•WRN migrates a Holliday junction with 3.2 ± 0.03 bases/s velocity•WRN reverses branch migration direction at short heterologous sequences•The branch migration reversal of WRN is an active process
By using single-molecule fluorescence measurements, Shin et al. show that the Holliday junction formation, branch migration, and migration direction reversal of WRN are all ATP dependent, revealing that WRN uses the energy of ATP hydrolysis to actively coordinate the structural transitions of DNA.
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.
Humans have five members of the well conserved RecQ helicase family: RecQ1, Bloom syndrome protein (BLM), Werner syndrome protein (WRN), RecQ4, and RecQ5, which are all known for their roles in ...maintaining genome stability. BLM, WRN, and RecQ4 are associated with premature aging and cancer predisposition. Of the three, RecQ4's biological and cellular roles have been least thoroughly characterized. Here we tested the helicase activity of purified human RecQ4 on various substrates. Consistent with recent results, we detected ATP-dependent RecQ4 unwinding of forked duplexes. However, our results provide the first evidence that human RecQ4's unwinding is independent of strand annealing, and that it does not require the presence of excess ssDNA. Moreover, we demonstrate that a point mutation of the conserved lysine in the Walker A motif abolished helicase activity, implying that not the N-terminal portion, but the helicase domain is solely responsible for the enzyme's unwinding activity. In addition, we demonstrate a novel stimulation of RecQ4's helicase activity by replication protein A, similar to that of RecQ1, BLM, WRN, and RecQ5. Together, these data indicate that specific biochemical activities and protein partners of RecQ4 are conserved with those of the other RecQ helicases.
Topoisomerases are essential for orderly nucleic acid metabolism and cell survival and are proven targets for clinically useful antimicrobial and anticancer drugs. Interest in the topologically ...intricate mitochondrial DNA (kinetoplast or kDNA) of Trypanosoma brucei brucei and related kinetoplastid protozoan parasites has led to many reports of type II topoisomerases that participate in kDNA metabolism (we term the T. brucei brucei gene TbTOP2mt). We have now identified and characterized two new genes for type II topoisomerases in T. brucei brucei, termed TbTOP2α and TbTOP2β. Phylogenetically, they share a common node with other nuclear topoisomerases, clearly distinct from a clade that includes the previously reported kinetoplastid genes, all of which are homologs of TbTOP2mt. Southern blot analysis reveals the new genes are single copy and positioned ∼1.7 kb apart. Cognate mRNAs are expressed in African trypanosomes, but only a single message is detected in Leishmania or Crithidia. TbTOP2α encodes an ATP-dependent topoisomerase that appears as a single ∼170-kDa band on immunoblots and localizes to the nucleus; RNA interference leads to pleomorphic nuclear (but not kDNA) abnormalities and early growth arrest. The role of TbTOP2β is unclear. Although transcribed in trypanosomes, TbTOP2β is not detected by β-specific antiserum, and RNAi silencing results in no obvious phenotype. These studies indicate that African trypanosomes and related kinetoplastid human pathogens are unusual in having independent topoisomerase II genes to service their nuclear and mitochondrial genomes, and they highlight TbTOP2α as a promising target for the development of much-needed new therapies.
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.
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► RAPADILINO syndrome is associated with mutations in RECQL4. ► We characterize RAPADILINO RECQL4's catalytic activities relative to WT RECQL4. ► We show that RAPADILINO RECQL4 lacks helicase and ssDNA-dependent ATPase activity. ► RAPADILINO and RTS patients may develop cancer due to loss of helicase activity.
Abstract
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.
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