RecQ Research Articles

HERC2 Facilitates BLM and WRN Helicase Complex Interaction with RPA to Suppress G-Quadruplex DNA.

BLM and WRN are RecQ DNA helicasesessential for genomic stability. Here, we demonstrate that HERC2, a HECT E3 ligase, is critical for their functions to suppress G-quadruplex (G4) DNA. HERC2 interacted with BLM, WRN, and replication protein A (RPA) complexes during the S-phase of the cell cycle. Depletion of HERC2 dissociated RPA from BLM and WRN complexes and significantly increased G4 formation. Triple depletion revealed that HERC2 has an epistatic relationship with BLM and WRN in their G4-suppressing function. In vitro, HERC2 released RPA onto single-stranded DNA (ssDNA) rather than anchoring onto RPA-coated ssDNA. CRISPR/Cas9-mediated deletion of the catalytic ubiquitin-binding site of HERC2 inhibited ubiquitination of RPA2, caused RPA accumulation in the helicase complexes, and increased G4, indicating an essential role for E3 activity in the suppression of G4. Both depletion of HERC2 and inactivation of E3 sensitized cells to the G4-interacting compounds telomestatin and pyridostatin. Overall, these results indicate that HERC2 is a master regulator of G4 suppression that affects the sensitivity of cells to G4 stabilizers. Given that HERC2 expression is frequently reduced in many types of cancers, G4 accumulation as a result of HERC2 deficiency may provide a therapeutic target for G4 stabilizers.Significance: HERC2 is revealed as a master regulator of G-quadruplex, a DNA secondary structure that triggers genomic instability and may serve as a potential molecular target in cancer therapy.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/22/6371/F1.large.jpg Cancer Res; 78(22); 6371-85. ©2018 AACR.

Spore-autonomous fluorescent protein expression identifies meiotic chromosome mis-segregation as the principal cause of hybrid sterility in yeast.

Genome-wide sequence divergence between populations can cause hybrid sterility through the action of the anti-recombination system, which rejects crossover repair of double strand breaks between nonidentical sequences. Because crossovers are necessary to ensure proper segregation of homologous chromosomes during meiosis, the reduced recombination rate in hybrids can result in high levels of nondisjunction and therefore low gamete viability. Hybrid sterility in interspecific crosses of Saccharomyces yeasts is known to be associated with such segregation errors, but estimates of the importance of nondisjunction to postzygotic reproductive isolation have been hampered by difficulties in accurately measuring nondisjunction frequencies. Here, we use spore-autonomous fluorescent protein expression to quantify nondisjunction in both interspecific and intraspecific yeast hybrids. We show that segregation is near random in interspecific hybrids. The observed rates of nondisjunction can explain most of the sterility observed in interspecific hybrids through the failure of gametes to inherit at least one copy of each chromosome. Partially impairing the anti-recombination system by preventing expression of the RecQ helicase SGS1 during meiosis cuts nondisjunction frequencies in half. We further show that chromosome loss through nondisjunction can explain nearly all of the sterility observed in hybrids formed between two populations of a single species. The rate of meiotic nondisjunction of each homologous pair was negatively correlated with chromosome size in these intraspecific hybrids. Our results demonstrate that sequence divergence is not only associated with the sterility of hybrids formed between distantly related species but may also be a direct cause of reproductive isolation in incipient species.

Werner Syndrome Helicase Is Required for the Survival of Cancer Cells with Microsatellite Instability

Werner Syndrome protein (WRN) is a RecQ enzyme involved in the maintenance of genome integrity. Germline loss of function mutations in WRN lead to premature aging and pre-disposition to cancer. We evaluated synthetic lethality (SL) between not only of WRN, but also of another human RecQ helicase, BLM, with DNA damage response genes in cancer cell lines. We found that WRN exhibited SL with a DNA mismatch repair protein MutL homolog 1 (MLH1), loss of which is associated with high microsatellite instability (MSI-H). We then demonstrated that MSI-H cells exhibited increased double-stranded DNA breaks, altered cell cycles and decreased viability in response to WRN knockdown. Although WRN is the only human RecQ enzyme with a distinct exonuclease domain, only loss of helicase activity drives the MSI SL interaction. This SL interaction in MSI cancer cells positions WRN as a relevant therapeutic target in patients with MSI-H tumors.

A guanine-flipping and sequestration mechanism for G-quadruplex unwinding by RecQ helicases

Homeostatic regulation of G-quadruplexes (G4s), four-stranded structures that can form in guanine-rich nucleic acids, requires G4 unwinding helicases. The mechanisms that mediate G4 unwinding remain unknown. We report the structure of a bacterial RecQ DNA helicase bound to resolved G4 DNA. Unexpectedly, a guanine base from the unwound G4 is sequestered within a guanine-specific binding pocket. Disruption of the pocket in RecQ blocks G4 unwinding, but not G4 binding or duplex DNA unwinding, indicating its essential role in structure-specific G4 resolution. A novel guanine-flipping and sequestration model that may be applicable to other G4-resolving helicases emerges from these studies.

Backbone Dynamics and Model‐Free Analysis of the RecQ C‐Terminal Domain of Bloom Syndrome Protein

Bloom syndrome protein (BLM) is one of RecQ helicases, which are conserved DNA unwinding enzymes in DNA metabolisms. The RecQ C‐terminal domain (RQC) of BLM is a winged‐helix motif, which recognizes various DNA structures. BLM RQC mediates electrostatic interactions with duplex DNA by using polar residues in the β‐wing and α2–α3 loop. In order to understand the dynamic properties of RQC for recognizing its substrate DNA, we measured 15N spin relaxation parameters of the protein and performed model‐free analysis. Our results showed that the β‐wing region, which is used for the strand separation, has flexible backbone structure. The α2–α3 loop, the binding surface of the DNA major groove, was found to be relatively rigid. Our results contribute to a better understanding of molecular basis of BLM RQC–DNA interaction.

RecQ helicases in the malaria parasite Plasmodium falciparum affect genome stability, gene expression patterns and DNA replication dynamics.

The malaria parasite Plasmodium falciparum has evolved an unusual genome structure. The majority of the genome is relatively stable, with mutation rates similar to most eukaryotic species. However, some regions are very unstable with high recombination rates, driving the generation of new immune evasion-associated var genes. The molecular factors controlling the inconsistent stability of this genome are not known. Here we studied the roles of the two putative RecQ helicases in P. falciparum, PfBLM and PfWRN. When PfWRN was knocked down, recombination rates increased four-fold, generating chromosomal abnormalities, a high rate of chimeric var genes and many microindels, particularly in known ‘fragile sites’. This is the first identification of a gene involved in suppressing recombination and maintaining genome stability in Plasmodium. By contrast, no change in mutation rate appeared when the second RecQ helicase, PfBLM, was mutated. At the transcriptional level, however, both helicases evidently modulate the transcription of large cohorts of genes, with several hundred genes—including a large proportion of vars—showing deregulated expression in each RecQ mutant. Aberrant processing of stalled replication forks is a possible mechanism underlying elevated mutation rates and this was assessed by measuring DNA replication dynamics in the RecQ mutant lines. Replication forks moved slowly and stalled at elevated rates in both mutants, confirming that RecQ helicases are required for efficient DNA replication. Overall, this work identifies the Plasmodium RecQ helicases as major players in DNA replication, antigenic diversification and genome stability in the most lethal human malaria parasite, with important implications for genome evolution in this pathogen.

Abstract 1245: Identification of germline variants in cancer susceptibility genes in patients with hereditary breast and ovarian cancer syndrome by massive parallel sequencing in Argentinean population

Abstract Breast cancer is the most frequent cancer and ovarian cancer the seventh most frequent cancer among females worldwide, representing approximately 25% and 4% of all cancers. In Argentina, breast cancer and Ovarian cancer cause 6163 and 1332 deaths per year, respectively. Both breast and ovarian cancers are heterogeneous diseases composed of different tumor types with distinctive features and behaviors. The main risk factors for breast and ovarian cancer include age, family history, and genetics.The genetic components of both of the diseases have been well established, contributing to up to 10% of all breast cancer cases and 15% of all ovarian cancer cases.The two major susceptibility genes for both diseases are BRCA1 and BRCA2, and several other susceptibility genes have been identified. However, in the majority of high-risk breast and/or ovarian cancer (HBOC) families, the genetic predisposition factors remain unidentified, making the genetic counseling of these families challenging. The aim of the current study is to utilize an NGS approach to identify genetic factors that predispose individuals to hereditary breast and/or ovarian cancer (HBOC) in the high-risk Argentinian BRCA1/2 mutation-negative HBOC patients.16 patients were analyzed, the DNA was extracted from a blood samples and was subjected to a targeted massively parallel sequencing platform comprising 141 cancer predisposition genes. We detected in these patient 6 pathogenic variants in SBDS, FANCA, MC1R, WRN, MSR1 and SDHD genes. The effect of these variants are of type: non-synonymous (67%) and stop codon gains (33%). We also found 6 variants of uncertain significance (VUS) in APC, RAD50, SDHB, FANCD2, MLH1 and CDH1 genes.In conclusion, the identification of new variants could help to discover new genes associated to HBOC in the population of Argentina.These findings provide an excellent premise for further studies and could be utilized in the design of more efficient clinical management strategies for HBOC. Citation Format: Alejandra Franco, Fernando Orti, Sandra Perdomo, Cecilia Riggi, Cecilia Frecha, Javier Oliver. Identification of germline variants in cancer susceptibility genes in patients with hereditary breast and ovarian cancer syndrome by massive parallel sequencing in Argentinean population [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1245.

Abstract 330: BRCA1 and BLM helicase compensate for replication fork defects in the absence of 53BP1 protein

Abstract 53BP1 is well-known for its involvement in DNA double strand break (DSB) repair, binding DNA and influencing choice of DSB repair. In S/G2 phases of the cell cycle, 53BP1 accumulates at DSBs, but can be removed by BRCA1, allowing end-resection and homology-directed repair (HDR) to occur. Without BRCA1, HDR is defective unless 53BP1 is also lost, in which case HDR can proceed. Such secondary mutation is relevant as a mechanism of acquired chemoresistance in BRCA1 deficient tumors. BRCA1 is also important for maintaining replication fork stability, particular for blocking end resection by MRE11a at a collapsed fork. We therefore set out to ask whether 53BP1 also plays a role in maintenance of fork stability and how this role relates to the role of BRCA1 at a collapsed fork. 53BP1 has been indicated to co-localize with collapsed forks and indeed we find that in its absence fork stability is compromised in a DNA2, but not MRE11a, dependent manner. With decreased fork stability in the absence of 53BP1 we observe increased HDR, indicating a role for BRCA1 to compensate for the lack of 53BP1 by promoting HDR. However, if both 53BP1 and BRCA1 are both necessary for fork stability, then there must be additional compensating mechanisms providing fork stability when both are absent. In fact, absence of both 53BP1 and BRCA1 results in better replication progression than when either 53BP1 or BRCA1 alone are absent. A third mechanism of fork stabilization was identified involving BLM; a RecQ helicase involved in dissolving homologous recombination intermediates, can facilitate fork restart and reverse fork regression. Additionally, BLM has been shown to interact with both 53BP1 and BRCA1, is found at stalled replication forks, and promotes BRCA1 recruitment to sites of DNA damage. Targeting BLM in cells with impaired 53BP1 and BRCA1 increases chemosensitivity to DNA damaging agents, supporting the redundant nature of these three proteins at stalled replication forks. Our findings indicate that targeting BLM may be an effective treatment for HDR defective cancers that have developed chemoresistance; with applicability to BRCA1 and homologous recombination deficient cancers, such as breast, ovarian, and Ewing sarcoma. Citation Format: Erin H. Sybouts, Sonal S. Tonapi, Alexander J. Bishop. BRCA1 and BLM helicase compensate for replication fork defects in the absence of 53BP1 protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 330.

Nonfunctional mutant Wrn protein leads to neurological deficits, neuronal stress, microglial alteration, and immune imbalance in a mouse model of Werner syndrome

Werner syndrome (WS) is a premature aging disorder caused by mutations in a RecQ-family DNA helicase, WRN. Mice lacking part of the helicase domain of the WRN orthologue exhibit many phenotypic features of WS, including metabolic abnormalities and a shorter lifespan. Yet, little is known about the impact of WRN mutations on the central nervous system in both humans and mouse models of WS. In the current study, we have performed a longitudinal behavioral assessment on mice bearing a Wrn helicase deletion. Behavioral tests demonstrated a loss of motor activity and coordination, reduction in perception, increase in repetitive behavior, and deficits in both spatial and social novelty memories in Wrn mutant mice compared to age-matched wild type mice. These neurological deficits were associated with biochemical and histological changes in the brain of aged Wrn mutant mice. Microglia, resident immune cells that regulate neuronal plasticity and function in the brain, were hyper-ramified in multiple regions involved with the behavioral deficits of Wrn mutant mice. Furthermore, western analyses indicated that Wrn mutant mice exhibited an increase of oxidative stress markers in the prefrontal cortex. Supporting these findings, electron microscopy studies revealed increased cellular aging and oxidative stress features, among microglia and neurons respectively, in the prefrontal cortex of aged Wrn mutant mice. In addition, multiplex immunoassay of serum identified significant changes in the expression levels of several pro- and anti-inflammatory cytokines. Taken together, these findings indicate that microglial dysfunction and neuronal oxidative stress, associated with peripheral immune system alterations, might be important driving forces leading to abnormal neurological symptoms in WS thus suggesting potential therapeutic targets for interventions.

Rev7 and 53BP1/Crb2 prevent RecQ helicase-dependent hyper-resection of DNA double-strand breaks.

Poly(ADP ribose) polymerase inhibitors (PARPi) target cancer cells deficient in homology-directed repair of DNA double-strand breaks (DSBs). In preclinical models, PARPi resistance is tied to altered nucleolytic processing (resection) at the 5' ends of a DSB. For example, loss of either 53BP1 or Rev7/MAD2L2/FANCV derepresses resection to drive PARPi resistance, although the mechanisms are poorly understood. Long-range resection can be catalyzed by two machineries: the exonuclease Exo1, or the combination of a RecQ helicase and Dna2. Here, we develop a single-cell microscopy assay that allows the distinct phases and machineries of resection to be interrogated simultaneously in living S. pombe cells. Using this assay, we find that the 53BP1 orthologue and Rev7 specifically repress long-range resection through the RecQ helicase-dependent pathway, thereby preventing hyper-resection. These results suggest that 'rewiring' of BRCA1-deficient cells to employ an Exo1-independent hyper-resection pathway is a driver of PARPi resistance.

RecQ4 helicases stimulate nuclease activity during DNA inter‐strand crosslink repair.

Mutations in the RecQ4 DNA helicase cause genomic instability that leads to a predisposition to various cancers and/or premature aging. Despite its importance to human health, however, RecQ4 remains the least studied of the five human RecQ helicases because it is difficult to work with both in vivo and in vitro. We recently characterized a putative RecQ4 homolog from Saccharomyces cerevisiae called Hrq1 and found that like RecQ4, it is involved in telomere maintenance and DNA inter‐strand crosslink (ICL) repair. Our genetic, biochemical, and structural data indicate that Hrq1 is a bona fide RecQ4 homolog and represents a simple model to study the roles of RecQ4 in genome integrity. Currently, we are focusing on Hrq1's mechanism of action during ICL repair. S. cerevisiae cells lacking the HRQ1 gene or expressing a catalytically inactive allele (hrq1‐K318A) are highly sensitive to the ICL‐inducing drugs such as mitomycin C, and this sensitivity is epistatic to mutations in the nuclease Pso2, which defines the major ICL repair pathway in yeast. In contrast, Hrq1 does not function in the proto‐Fanconi Anemia pathway in S. cerevisiae. By analogy to another well characterized RecQ helicase/nuclease pair in yeast that is responsible for end resection during double‐strand break repair (Sgs1/Dna2), we hypothesized that Hrq1 may physically interact with Pso2 to facilitate ICL repair. Using purified proteins, we found that Hrq1 stimulates Pso2 nuclease activity on short DNA substrates. Further, reciprocal pull‐down experiments indicate that Hrq1 and Pso2 physically associate, suggesting that nuclease stimulation is direct rather than the result of Hrq1 generating a preferred substrate for Pso2. Similar in vitro nuclease stimulation is achieved with recombinant RecQ4 but not with S. cerevisiae Sgs1, suggesting that it is specific to RecQ4 family helicases. Experiments are now underway using bacterial and archaeal RecQ4 homologs to determine if this interaction is evolutionarily conserved among all domains of life and to identify the interaction motifs on the proteins.Support or Funding InformationSupport was provided by the College of Arts and Sciences, Indiana University, an Indiana University Collaborative Research Grant from the Office of the Vice President for Research, and the American Cancer Society [RSG‐16‐180‐01‐DMC].This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Catalytic functions of RECQ1 helicase are important in defying genomic stress and maintaining cell proliferation

RecQ helicases are an important class of DNA repair proteins with critical roles in genome maintenance. RECQ1, a structure‐specific DNA helicase and the most abundant RecQ homolog in humans, is frequently upregulated in rapidly proliferating cells and has dual roles in genome maintenance and transcription regulation. RECQ1's function as well as expression levels modulates patient survival and response to anti‐cancer therapy. More recently germline mutations in RECQ1 have been linked to breast cancer susceptibility. However, the mechanism by which altered RECQ1 expression or mutations in functional domains of RECQ1 modulate response to chemotherapy and increase cancer risk is unclear. Our study aimed at generating a RECQ1 knockout cell line using CRISPR‐Cas9 technology to investigate phenotypes of cells that lack RECQ1 expression completely or express breast cancer risk associated RECQ1 mutants in an isogenic background. Our results demonstrate that we have successfully generated a human RECQ1 knockout MDA‐MB‐231 breast cancer cell line using the CRIPSR‐Cas9 technique which recapitulates previously established phenotypes of RECQ1 knockdown resulting in reduced cell proliferation, growth and migration as well as increased sensitivity to camptothecin. We further demonstrate that the missense mutations in the evolutionarily conserved residues within the catalytic core of RECQ1 that predispose individuals to breast cancer, also lead to impaired cell proliferation, increased sensitivity to replication stress and accumulation of spontaneous DNA damage indicating that defective helicase functions of RECQ1 may eventually contribute to genomic instability and drive cancer progression.Support or Funding InformationThis work was funded by the NIGMS/NIH grant SC1GM093999 to Sudha SharmaThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The RecQ‐like helicase HRQ1 is involved in DNA crosslink repair in Arabidopsis in a common pathway with the Fanconi anemia‐associated nuclease FAN1 and the postreplicative repair ATPase RAD5A

RecQ helicases are important caretakers of genome stability and occur in varying copy numbers in different eukaryotes. Subsets of RecQ paralogs are involved in DNA crosslink (CL) repair. The orthologs of AtRECQ2, AtRECQ3 and AtHRQ1, HsWRN, DmRECQ5 and ScHRQ1 participate in CL repair in their respective organisms, and we aimed to define the function of these helicases for plants. We obtained Arabidopsis mutants of the three RecQ helicases and determined their sensitivity against CL agents in single- and double-mutant analyses. Only Athrq1, but not Atrecq2 and Atrecq3, mutants proved to be sensitive to intra- and interstrand crosslinking agents. AtHRQ1 is specifically involved in the repair of replicative damage induced by CL agents. It shares pathways with the Fanconi anemia-related endonuclease FAN1 but not with the endonuclease MUS81. Most surprisingly, AtHRQ1 is epistatic to the ATPase RAD5A for intra- as well as interstrand CL repair. We conclude that, as in fungi, AtHRQ1 has a conserved function in DNA excision repair. Additionally, HRQ1 not only shares pathways with the Fanconi anemia repair factors, but in contrast to fungi also seems to act in a common pathway with postreplicative DNA repair.

RecQ Helicases Function in Development, DNA Repair, and Gene Targeting in Physcomitrella patens.

RecQ DNA helicases are genome surveillance proteins found in all kingdoms of life. They are characterized best in humans, as mutations in RecQ genes lead to developmental abnormalities and diseases. To better understand RecQ functions in plants we concentrated on Arabidopsis thaliana and Physcomitrella patens, the model species predominantly used for studies on DNA repair and gene targeting. Phylogenetic analysis of the six P. patens RecQ genes revealed their orthologs in humans and plants. Because Arabidopsis and P. patens differ in their RecQ4 and RecQ6 genes, reporter and deletion moss mutants were generated and gene functions studied in reciprocal cross-species and cross-kingdom approaches. Both proteins can be found in meristematic moss tissues, although at low levels and with distinct expression patterns. PpRecQ4 is involved in embryogenesis and in subsequent development as demonstrated by sterility of ΔPpRecQ4 mutants and by morphological aberrations. Additionally, ΔPpRecQ4 displays an increased sensitivity to DNA damages and an increased rate of gene targeting. Therefore, we conclude that PpRecQ4 acts as a repressor of recombination. In contrast, PpRecQ6 is not obviously important for moss development or DNA repair but does function as a potent enhancer of gene targeting.

Differential stem cell aging kinetics in Hutchinson-Gilford progeria syndrome and Werner syndrome

Hutchinson-Gilford progeria syndrome (HGPS) and Werner syndrome (WS) are two of the best characterized human progeroid syndromes. HGPS is caused by a point mutation in lamin A (LMNA) gene, resulting in the production of a truncated protein product—progerin. WS is caused by mutations in WRN gene, encoding a loss-of-function RecQ DNA helicase. Here, by gene editing we created isogenic human embryonic stem cells (ESCs) with heterozygous (G608G/+) or homozygous (G608G/G608G) LMNA mutation and biallelic WRN knockout, for modeling HGPS and WS pathogenesis, respectively. While ESCs and endothelial cells (ECs) did not present any features of premature senescence, HGPS- and WS-mesenchymal stem cells (MSCs) showed aging-associated phenotypes with different kinetics. WS-MSCs had early-onset mild premature aging phenotypes while HGPS-MSCs exhibited late-onset acute premature aging characterisitcs. Taken together, our study compares and contrasts the distinct pathologies underpinning the two premature aging disorders, and provides reliable stem-cell based models to identify new therapeutic strategies for pathological and physiological aging.

When Helicase and Polymerases Collides and Unfolds G4-Quadruplex on their Track

Using single molecule technique, we first characterize the kinetics of folding and unfolding as well as the stability of a single G4 inserted in a dsDNA: a situation that mimics the G4s in promoters, where the complementary sequence competes with the G-rich structure. We use the fact that a DNA fork rezipping a molecule is arrested by the G4. We find that the lifetime of a single telomeric G4 is small (∼20s) and thus this G4 most likely unfolds before a helicase can reach it. This is not the case for the very stable c-MYC G4 which remains for typically ∼2h. For this G4, we observe in real time how helicases or polymerases behave as they collide the G4 on their track. We find that the Pif1 helicase resolves this G4 after marking a pause. On the other hand, this G4 is not resolved by the RecQ helicase nor by the bacteriophage T4 replicative helicase or the protein RPA, but it is unwound by the gp43 polymerase and by the T4 replisome. We also observe that the G4 may be jumped by those helicases which do not unfold the structure. We shall also discuss the case of other polymerases.

Interaction of replication protein A with two acidic peptides from human Bloom syndrome protein.

Bloom syndrome protein (BLM) is one of five human RecQ helicases which maintain genomic stability. Interaction of BLM with replication protein A (RPA) stimulates the DNA unwinding ability of BLM. The interaction is expected to be crucial in the DNA damage response. Although this stimulation of BLM by RPA is of particular importance in cancer cells, the precise binding surfaces of both proteins are not well understood. In this study, we show by fluorescence polarisation anisotropy that both acidic surface peptides of BLM specifically bind to the RPA70N domain of RPA. Our NMR analysis and docking models show that the basic cleft region of RPA70N is the binding site for both peptides and that the acidic peptide/basic cleft interaction governs RPA-BLM binding.

RECQ Helicase Triggers a Binding Mode Change in the SSB-DNA Complex Efficiently Initiate DNA Unwinding

RECQ Helicase Triggers a Binding Mode Change in the SSB-DNA Complex Efficiently Initiate DNA Unwinding

Folate modulates guanine-quadruplex frequency and DNA damage in Werner syndrome

Guanine-quadruplexes (G4) are stable tetra-stranded DNA structures that may cause DNA replication stress and inhibit gene expression. Defects in unwinding these structures by DNA helicases may result in telomere shortening and DNA damage. Furthermore, due to mutations in WRN helicase genes in Werner syndrome, G4 motifs are likely to be key elements in the expression of premature aging phenotypes. The methylation of DNA plays a significant role in the stability and occurrence of G4. Thus, G4 frequency and DNA methylation mechanisms may be affected by excesses or deficiencies in methyl donors such as folate. B-Lymphocytes from Werner patients (n = 5) and healthy individuals (n = 5) were cultured in RPMI medium under condition of folate deficiency (20 nM) or sufficiency (200 nM) for 14 days. Cells were fixed on microscope slides for immunofluorescent staining to measure G4 frequency and γH2AX (a marker of DNA strand breaks) intensity, using automated quantitative imaging fluorescent microscopy. There was a significant increase (p < 0.05) in G4 levels in Werner syndrome patients compared to healthy controls. Werner and control cells grown in 20 nM folate media also showed significant increases in G4 (p < 0.001) and γH2AX (p < 0.01) signals compared with the same cells grown in 200 nM folate. Control cells grown in 20 nM folate also showed a significant reduction in DNA methylation levels (P < 0.05). The results of this study suggest that the occurrence of DNA G4 structures can be modulated in vitro via nutrients with important roles in methylation.

Human RecQL4 helicase plays multifaceted roles in the genomic stability of normal and cancer cells

Human RecQ helicases that share homology with E. coli RecQ helicase play critical roles in diverse biological activities such as DNA replication, transcription, recombination and repair. Mutations in three of the five human RecQ helicases (RecQ1, WRN, BLM, RecQL4 and RecQ5) result in autosomal recessive syndromes characterized by accelerated aging symptoms and cancer incidence. Mutational inactivation of Werner (WRN) and Bloom (BLM) genes results in Werner syndrome (WS) and Bloom syndrome (BS) respectively. However, mutations in RecQL4 result in three human disorders: (I) Rothmund-Thomson syndrome (RTS), (II) RAPADILINO and (III) Baller-Gerold syndrome (BGS). Cells from WS, BS and RTS are characterized by a unique chromosomal anomaly indicating that each of the RecQ helicases performs specialized function(s) in a non-redundant manner. Elucidating the biological functions of RecQ helicases will enable us to understand not only the aging process but also to determine the cause for age-associated human diseases. Recent biochemical and molecular studies have given new insights into the multifaceted roles of RecQL4 that range from genomic stability to carcinogenesis and beyond. This review summarizes some of the existing and emerging knowledge on diverse biological functions of RecQL4 and its significance as a potential molecular target for cancer therapy.