ssDNA reeling is an intermediate step in the reiterative DNA unwinding activity of the WRN-1 helicase.

RecQ helicases are highly conserved between bacteria and humans. These helicases unwind various DNA structures in the 3' to 5'. Defective helicase activity elevates genomic instability and is associated with predisposition to cancer and/or premature aging. Recent single-molecule analyses have revealed the repetitive unwinding behavior of RecQ helicases from Escherichia coli to humans. However, the detailed mechanisms underlying this behavior are unclear. Here, we performed single-molecule studies of WRN-1 Caenorhabditis elegans RecQ helicase on various DNA constructs and characterized WRN-1 unwinding dynamics. We showed that WRN-1 persistently repeated cycles of DNA unwinding and rewinding with an unwinding limit of 25 to 31 bp per cycle. Furthermore, by monitoring the ends of the displaced strand during DNA unwinding we demonstrated that WRN-1 reels in the ssDNA overhang in an ATP-dependent manner. While WRN-1 reeling activity was inhibited by a C. elegans homolog of human replication protein A, we found that C. elegans replication protein A actually switched the reiterative unwinding activity of WRN-1 to unidirectional unwinding. These results reveal that reeling-in ssDNA is an intermediate step in the reiterative unwinding process for WRN-1 (i.e., the process proceeds via unwinding-reeling-rewinding). We propose that the reiterative unwinding activity of WRN-1 may prevent extensive unwinding, allow time for partner proteins to assemble on the active region, and permit additional modulation in vivo.

Molecular characteristics of reiterative DNA unwinding by the Caenorhabditis elegans RecQ helicase.

The RecQ family of helicases is highly conserved both structurally and functionally from bacteria to humans. Defects in human RecQ helicases are associated with genetic diseases that are characterized by cancer predisposition and/or premature aging. RecQ proteins exhibit 3'-5' helicase activity and play critical roles in genome maintenance. Recent advances in single-molecule techniques have revealed the reiterative unwinding behavior of RecQ helicases. However, the molecular mechanisms involved in this process remain unclear, with contradicting reports. Here, we characterized the unwinding dynamics of the Caenorhabditis elegans RecQ helicase HIM-6 using single-molecule fluorescence resonance energy transfer measurements. We found that HIM-6 exhibits reiterative DNA unwinding and the length of DNA unwound by the helicase is sharply defined at 25-31 bp. Experiments using various DNA substrates revealed that HIM-6 utilizes the mode of 'sliding back' on the translocated strand, without strand-switching for rewinding. Furthermore, we found that Caenorhabditis elegans replication protein A, a single-stranded DNA binding protein, suppresses the reiterative behavior of HIM-6 and induces unidirectional, processive unwinding, possibly through a direct interaction between the proteins. Our findings shed new light on the mechanism of DNA unwinding by RecQ family helicases and their co-operation with RPA in processing DNA.

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.

The Caenorhabditis elegans Werner syndrome protein functions upstream of ATR and ATM in response to DNA replication inhibition and double-strand DNA breaks.

WRN-1 is the Caenorhabditis elegans homolog of the human Werner syndrome protein, a RecQ helicase, mutations of which are associated with premature aging and increased genome instability. Relatively little is known as to how WRN-1 functions in DNA repair and DNA damage signaling. Here, we take advantage of the genetic and cytological approaches in C. elegans to dissect the epistatic relationship of WRN-1 in various DNA damage checkpoint pathways. We found that WRN-1 is required for CHK1 phosphorylation induced by DNA replication inhibition, but not by UV radiation. Furthermore, WRN-1 influences the RPA-1 focus formation, suggesting that WRN-1 functions in the same step or upstream of RPA-1 in the DNA replication checkpoint pathway. In response to ionizing radiation, RPA-1 focus formation and nuclear localization of ATM depend on WRN-1 and MRE-11. We conclude that C. elegans WRN-1 participates in the initial stages of checkpoint activation induced by DNA replication inhibition and ionizing radiation. These functions of WRN-1 in upstream DNA damage signaling are likely to be conserved, but might be cryptic in human systems due to functional redundancy.

Physical and functional interactions of Caenorhabditis elegans WRN-1 helicase with RPA-1.

The Caenorhabditis elegans Werner syndrome protein, WRN-1, a member of the RecQ helicase family, has a 3'-5' DNA helicase activity. Worms with defective wrn-1 exhibit premature aging phenotypes and an increased level of genome instability. In response to DNA damage, WRN-1 participates in the initial stages of checkpoint activation in concert with C. elegans replication protein A (RPA-1). WRN-1 helicase is stimulated by RPA-1 on long DNA duplex substrates. However, the mechanism by which RPA-1 stimulates DNA unwinding and the function of the WRN-1-RPA-1 interaction are not clearly understood. We have found that WRN-1 physically interacts with two RPA-1 subunits, CeRPA73 and CeRPA32; however, full-length WRN-1 helicase activity is stimulated by only the CeRPA73 subunit, while the WRN-1(162-1056) fragment that harbors the helicase activity requires both the CeRPA73 and CeRPA32 subunits for the stimulation. We also found that the CeRPA73(1-464) fragment can stimulate WRN-1 helicase activity and that residues 335-464 of CeRPA73 are important for physical interaction with WRN-1. Because CeRPA73 and the CeRPA73(1-464) fragment are able to bind single-stranded DNA (ssDNA), the stimulation of WRN-1 helicase by RPA-1 is most likely due to the ssDNA binding activity of CeRPA73 and the direct interaction of WRN-1 and CeRPA73.

The structure of the trimer of human 4-1BB ligand is unique among members of the tumor necrosis factor superfamily.

Binding of the 4-1BB ligand (4-1BBL) to its receptor, 4-1BB, provides the T lymphocyte with co-stimulatory signals for survival, proliferation, and differentiation. Importantly, the 4-1BB-4-1BBL pathway is a well known target for anti-cancer immunotherapy. Here we present the 2.3-A crystal structure of the extracellular domain of human 4-1BBL. The ectodomain forms a homotrimer with an extended, three-bladed propeller structure that differs from trimers formed by other members of the tumor necrosis factor (TNF) superfamily. Based on the 4-1BBL structure, we modeled its complex with 4-1BB, which was consistent with images obtained by electron microscopy, and verified the binding site by site-directed mutagenesis. This structural information will facilitate the development of immunotherapeutics targeting 4-1BB.

DNA secondary structure of the released strand stimulates WRN helicase action on forked duplexes without coordinate action of WRN exonuclease.

Werner syndrome (WS) is an autosomal recessive premature aging disorder characterized by aging-related phenotypes and genomic instability. WS is caused by mutations in a gene encoding a nuclear protein, Werner syndrome protein (WRN), a member of the RecQ helicase family, that interestingly possesses both helicase and exonuclease activities. Previous studies have shown that the two activities act in concert on a single substrate. We investigated the effect of a DNA secondary structure on the two WRN activities and found that a DNA secondary structure of the displaced strand during unwinding stimulates WRN helicase without coordinate action of WRN exonuclease. These results imply that WRN helicase and exonuclease activities can act independently, and we propose that the uncoordinated action may be relevant to the in vivo activity of WRN.

Longevity and resistance to stress correlate with DNA repair capacity in Caenorhabditis elegans.

DNA repair is an important mechanism by which cells maintain genomic integrity. Decline in DNA repair capacity or defects in repair factors are thought to contribute to premature aging in mammals. The nematode Caenorhabditis elegans is a good model for studying longevity and DNA repair because of key advances in understanding the genetics of aging in this organism. Long-lived C. elegans mutants have been identified and shown to be resistant to oxidizing agents and UV irradiation, suggesting a genetically determined correlation between DNA repair capacity and life span. In this report, gene-specific DNA repair is compared in wild-type C. elegans and stress-resistant C. elegans mutants for the first time. DNA repair capacity is higher in long-lived C. elegans mutants than in wild-type animals. In addition, RNAi knockdown of the nucleotide excision repair gene xpa-1 increased sensitivity to UV and reduced the life span of long-lived C. elegans mutants. These findings support that DNA repair capacity correlates with longevity in C. elegans.

Biochemical characterization of the WRN-1 RecQ helicase of Caenorhabditis elegans.

The highly conserved RecQ helicases are essential for the maintenance of genomic stability. Werner syndrome protein, WRN, is one of five human RecQ helicase homologues, and a deficiency of the protein causes a hereditary premature aging disorder that is characterized by genomic instability. A WRN orthologue, wrn-1 lacking the exonuclease domain, has been identified in the nematode Caenorhabditis elegans. wrn-1(RNAi) in C. elegans has a shortened life span, increased sensitivity to DNA damage, and accelerated aging phenotypes. However, little is known about its enzymatic activity. We purified the recombinant C. elegans WRN-1 protein (CeWRN-1) and then investigated its substrate specificity in vitro to improve our understanding of its function in vivo. We found that CeWRN-1 is an ATP-dependent 3'-5' helicase capable of unwinding a variety of DNA structures such as forked duplexes, Holliday junctions, bubble substrates, D-loops, and flap duplexes, and 3'-tailed duplex substrates. Distinctly, CeWRN-1 is able to unwind a long forked duplex compared to human WRN. Furthermore, CeWRN-1 helicase activity on a long DNA duplex is stimulated by C. elegans replication protein A (CeRPA) that is shown to interact with CeWRN-1 by a dot blot. The ability of CeWRN-1 to unwind these DNA structures may improve the access for DNA repair and replication proteins that are important for preventing the accumulation of abnormal structures, contributing to genomic stability.

Crystallization and preliminary X-ray crystallographic study of the extracellular domain of the 4-1BB ligand, a member of the TNF family.

The 4-1BB ligand, a member of the tumour necrosis factor (TNF) family, is an important co-stimulatory molecule that plays a key role in the clonal expansion and survival of CD8+ T cells. Signalling through binding of the 4-1BB ligand and 4-1BB has been reported to enhance CD8+ T-cell expansion and protect activated CD8+ T cells from death. The 4-1BB ligand is an integral protein expressed on activated antigen-presenting cells. The extracellular domain of the 4-1BB ligand fused with glutathione-S-transferase was expressed in Escherichia coli (Origami) and purified by using affinity and ion-exchange column chromatographic methods. Crystals of the 4-1BB ligand were obtained at 290 K by the hanging-drop vapour-diffusion method. X-ray diffraction data were collected from these crystals to 2.8 A resolution and the crystals belong to space group C2, with unit-cell parameters a = 114.6, b = 73.8, c = 118.50 A, beta = 115.5 degrees.

DNA Strand Breaks in Mitotic Germ Cells of Caenorhabditis elegans Evaluated by Comet Assay.

DNA damage responses are important for the maintenance of genome stability and the survival of organisms. Such responses are activated in the presence of DNA damage and lead to cell cycle arrest, apoptosis, and DNA repair. In Caenorhabditis elegans, double-strand breaks induced by DNA damaging agents have been detected indirectly by antibodies against DSB recognizing proteins. In this study we used a comet assay to detect DNA strand breaks and to measure the elimination of DNA strand breaks in mitotic germline nuclei of C. elegans. We found that C. elegans brc-1 mutants were more sensitive to ionizing radiation and camptothecin than the N2 wild-type strain and repaired DNA strand breaks less efficiently than N2. This study is the first demonstration of direct measurement of DNA strand breaks in mitotic germline nuclei of C. elegans. This newly developed assay can be applied to detect DNA strand breaks in different C. elegans mutants that are sensitive to DNA damaging agents.

The Caenorhabditis elegans Werner syndrome protein participates in DNA damage checkpoint and DNA repair in response to CPT-induced double-strand breaks.

The RecQ helicases play roles in maintenance of genomic stability in species ranging from Escherichia coli to humans and interact with proteins involved in DNA metabolic pathways such as DNA repair, recombination, and replication. Our previous studies found that the Caenorhabditis elegans WRN-1 RecQ protein (a human WRN ortholog) exhibits ATP-dependent 3'-5' helicase activity and that the WRN-1 helicase is stimulated by RPA-1 on a long forked DNA duplex. However, the role of WRN-1 in response to S-phase associated with DSBs is unclear. We found that WRN-1 is involved in the checkpoint response to DSBs after CPT, inducing cell cycle arrest, is recruited to DSBs by RPA-1 and functions upstream of ATL-1 and ATM-1 for CHK-1 phosphorylation in the S-phase checkpoint. In addition, WRN-1 and RPA-1 recruitments to the DSBs require MRE-11, suggesting that DSB processing controlled by MRE-11 is important for WRN-1 at DSBs. The repair of CPT-induced DSBs is greatly reduced in the absence of WRN-1. These observations suggest that WRN-1 functions downstream of RPA-1 and upstream of CHK-1 in the DSB checkpoint pathway and is also required for the repair of DSB.

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.

A DNA repair gene of Caenorhabditis elegans: a homolog of human XPF.

The xeroderma pigmentosum complementation group F (XPF) protein is a structure-specific endonuclease in a complex with ERCC1 and is essential for nucleotide excision repair (NER). We report a single cDNA of Caenorhabditis elegans (C. elegans) encoding highly similar protein to human XPF and other XPF members. We propose to name the corresponding C. elegans gene xpf. Messenger RNA for C. elegans xpf is 5'-tagged with a SL2 splice leader, suggesting an operon-like expression for xpf. Using RNAi, we showed that loss of C. elegans xpf function caused hypersensitivity to ultra-violet (UV) irradiation, as observed in enhanced germ cell apoptosis and increased embryonic lethality. This study suggests that C. elegans xpf is conserved in evolution and plays a role in the repair of UV-damaged DNA in C. elegans.

Genetic polymorphisms of ataxia telangiectasia mutated and breast cancer risk.

To evaluate the role of genetic polymorphisms of ataxia telangiectasia mutated (ATM) in the etiology of breast cancer, a hospital-based case-control study was conducted in Korea. Nine-hundred ninety-six histologically confirmed incident breast cancer cases and 1,181 cancer-free controls were recruited in Seoul between 1995 and 2003. Genotypes of the ATM polymorphisms-5144A > T, IVS21 + 1049T > C, IVS33 - 55T > C, IVS34 + 60G > A, and 3393T > G were determined by the 5'-nuclease assay. Individual haplotypes were estimated from genotype data by a Bayesian method. Five ATM alleles were found to be in strong linkage disequilibrium (D' > 0.82; P < 0.001). Haplotype frequencies were significantly different between cases and controls (chi2 test, P < 0.001). The ATM IVS21 + 1049 TC or CC, IVS34 + 60 GA or AA, and 3393 TG or GG genotypes were associated with increased breast cancer risk, particularly in premenopausal women [odds ratios (OR), 1.51; 95% confidence interval (CI), 1.11-2.05; OR, 1.42; 95% CI, 1.08-1.88; and OR, 1.37; 95% CI, 1.04-1.80, respectively]. Compared with diploid of TCCAG:TCCAG, the most common haplotype, the ATTGT:ATTGT was associated with decreased risk of breast cancer with borderline significance (OR, 0.77; 95% CI, 0.58-1.04) and TCCAG:ATCGT and ATTGT:ACCAG were associated with increased breast cancer risk (OR, 2.30; 95% CI, 1.18-4.48 and OR, 2.43; 95% CI, 1.1.07-5.52, respectively) after adjusting for age, education, age at first full-term pregnancy, parity, family history of breast cancer, alcohol consumption, and smoking. As the number of ATTGT haplotype decreased, the risk of breast cancer increased (P for trend < 0.01). Our results thus suggest that genetic polymorphisms of ATM play an important role in the development of breast cancer in Korean women.

WRN translocation from nucleolus to nucleoplasm is regulated by SIRT1 and required for DNA repair and the development of chemoresistance.

When defective or absent, Werner syndrome protein (WRN) causes a genetic premature aging disorder called Werner syndrome. Several studies have reported that defects in WRN function are responsible for not only progeria syndrome but also genomic instability via the deregulation of DNA repair, replication, recombination, and telomere stability. Given the importance of WRN in the repair process, we herein investigated the potential role of WRN in drug response by evaluating the DNA repair following exposure to cisplatin in human cancer cell lines. We found that the down-regulation of SIRT1 and inhibition of SIRT1 deacetylase activity blocked the translocation of WRN from the nucleolus to the nucleoplasm in response to genotoxic stresses. In addition, cells expressing low levels of WRN responded favorably to cisplatin, whereas cells expressing high levels responded poorly to cisplatin. The forced expression of WRN protein in chemosensitive cells resulted in an approximately two-fold increase in cell viability in response to cisplatin compared with vector controls and promoted DNA repair, while WRN-deficient cells accumulate unrepaired double-strand breaks following cisplatin exposure. These results suggest that WRN is regulated by SIRT1 and increased expression of WRN might be one of the determinants for the development of chemotherapeutic drug resistance.

The gene expression and deficiency phenotypes of Cockayne syndrome B protein in Caenorhabditis elegans.

The Caenorhabditis elegans Cockayne syndrome B protein homologue is encoded by 10 exons of the predicted open reading frame F53H4.1. The gene is expressed in germ cells and all somatic cells of the embryonic to adult stage. Although the gene expression was ubiquitous, its expression level was relatively higher in dividing cells and cells that play fundamental roles in essential physiological functions such as feeding, sensation, and reproduction. RNA interference of the gene hypersensitized C. elegans to UV radiation, as observed in enhanced germ cell proliferation arrest and apoptosis, and increased embryonic lethality, suggesting its role in nucleotide excision repair.

Comparative binding of antitumor drugs to DNA containing the telomere repeat sequence.

Telomeres are the ends of the linear chromosomes of eukaryotes and consist of tandem GT-rich repeats in telomere sequence i.e. 500-3000 repeats of 5'-TTAGGG-3' in human somatic cells, which are shortened gradually with age. The G-rich overhang of telomere sequence can adopt different intramolecular fold-backs and tetra-stranded DNA structures, in vitro, which inhibit telomerase activity. In this report, DNA binding agents to telomere sequence were studied novel therapeutic possibility to destabilize telomeric DNA sequences. Oligonucleotides containing the guanine repeats in human telomere sequence were synthesized and used for screening potential antitumor drugs. Telomeric DNA sequence was characterized using spectral measurements and CD spectroscopy. CD spectrum indicated that the double-stranded telomeric DNA is in a right-handed conformation. Polyacrylamide gel electrophoresis was performed for binding behaviors of antitumor compounds with telomeric DNA sequence. Drugs interacted with DNA sequence caused changes in the electrophoretic mobility and band intensity of the gels. Depending on the binding mode of the anticancer drugs, telomeric DNA sequence was differently recognized and the efficiency of cleavage of DNA varies in the bleomycin-treated samples under different conditions. DNA cleavage occurred at about 1% by the increments of 1 micromM bleomycin-Fe(III). These results imply that the stability of human telomere sequence is important in conjunction with the cancer treatment and aging process.

Repair of mitomycin C cross-linked DNA in mammalian cells measured by a host cell reactivation assay.

DNA repair capacity in a cell could be detected by a host-cell reactivation assay (HCR). Since relation between DNA repair and genetic susceptibility to cancer remains unclear, it is necessary to identify DNA repair defects in human cancer cells. To assess DNA repair for breast cancer susceptibility, we developed a modified HCR assay using a plasmid containing a firefly luciferase gene damaged by mitomycin C (MMC), which forms interstrand cross-link (ICL) adducts. In particular, interstrand cross-link is thought to induce strand breaks being repaired by homologous recombination. The MMC-ICLs were verified by electrophoresis. Damaged plasmids were transfected into apparently normal human lymphocytes and NER-deficient XP cell lines and the DNA repair capacity of the cells were measured by quantifying the activity of the firefly luciferase. MMC lesion was repaired as much as UV adducts in normal lymphocytes and the XPC cells. However, the XPA cells have a lower repair capacity for MMC lesion than the XPC cell, indicating that the XPA protein may be involved in initial damage recognition of MMC-ICL adducts. Since several repair pathways including NER and recombination participate in MMC-ICL removal, this host cell reactivation assay using MMC-ICLs can be used in exploring DNA repair defects in human cancer cells.

Chromatin remodeling facilitates DNA incision in UV-damaged nucleosomes.

The DNA repair machinery must locate and repair DNA damage all over the genome. As nucleosomes inhibit DNA repair in vitro, it has been suggested that chromatin remodeling might be required for efficient repair in vivo. To investigate a possible contribution of nucleosome dynamics and chromatin remodeling to the repair of UV-photoproducts in nucleosomes, we examined the effect of a chromatin remodeling complex on the repair of UV-lesions by Micrococcus luteus UV endonuclease (ML-UV endo) and T4-endonuclease V (T4-endoV) in reconstituted mononucleosomes positioned at one end of a 175-bp long DNA fragment. Repair by ML-UV endo and T4-endoV was inefficient in mononucleosomes compared with naked DNA. However, the human nucleosome remodeling complex, hSWI/SNF, promoted more homogeneous repair by ML-UV endo and T4-endo V in reconstituted nucleosomes. This result suggests that recognition of DNA damage could be facilitated by a fluid state of the chromatin resulting from chromatin remodeling activities.