Interactions between the Werner syndrome helicase and DNA polymerase delta specifically facilitate copying of tetraplex and hairpin structures of the d(CGG)n trinucleotide repeat sequence.

Werner syndrome (WS) is an inherited disorder characterized by premature aging and genomic instability. The protein encoded by the WS gene, WRN, possesses intrinsic 3' --> 5' DNA helicase and 3' --> 5' DNA exonuclease activities. WRN helicase resolves alternate DNA structures including tetraplex and triplex DNA, and Holliday junctions. Thus, one function of WRN may be to unwind secondary structures that impede cellular DNA transactions. We report here that hairpin and G'2 bimolecular tetraplex structures of the fragile X expanded sequence, d(CGG)(n), effectively impede synthesis by three eukaryotic replicative DNA polymerases (pol): pol alpha, pol delta, and pol epsilon. The constraints imposed on pol delta-catalyzed synthesis are relieved, however, by WRN; WRN facilitates pol delta to traverse these template secondary structures to synthesize full-length DNA products. The alleviatory effect of WRN is limited to pol delta; neither pol alpha nor pol epsilon can traverse template d(CGG)(n) hairpin and tetraplex structures in the presence of WRN. Alleviation of pausing by pol delta is observed with Escherichia coli RecQ but not with UvrD helicase, suggesting a concerted action of RecQ helicases and pol delta. Our findings suggest a possible role of WRN in rescuing pol delta-mediated replication at forks stalled by unusual DNA secondary structures.

Functional interaction between the Werner Syndrome protein and DNA polymerase delta.

Werner Syndrome (WS) is an inherited disease characterized by premature onset of aging, increased cancer incidence, and genomic instability. The WS gene encodes a 1,432-amino acid polypeptide (WRN) with a central domain homologous to the RecQ family of DNA helicases. Purified WRN unwinds DNA with 3'-->5' polarity, and also possesses 3'-->5' exonuclease activity. Elucidation of the physiologic function(s) of WRN may be aided by the identification of WRN-interacting proteins. We show here that WRN functionally interacts with DNA polymerase delta (pol delta), a eukaryotic polymerase required for DNA replication and DNA repair. WRN increases the rate of nucleotide incorporation by pol delta in the absence of proliferating cell nuclear antigen (PCNA) but does not stimulate the activity of eukaryotic DNA polymerases alpha or epsilon, or a variety of other DNA polymerases. Moreover, we show that functional interaction with WRN is mediated through the third subunit of pol delta: i.e., Pol32p of Saccharomyces cerevisae, corresponding to the recently identified p66 subunit of human pol delta. Absence of the third subunit abrogates stimulation by WRN, and stimulation is restored by reconstituting the three-subunit enzyme. Our findings suggest that WRN may facilitate pol delta-mediated DNA replication and/or DNA repair and that disruption of WRN-pol delta interaction in WS cells may contribute to the previously observed S-phase defects and/or the unusual sensitivity to a limited number of DNA damaging agents.

WRN helicase expression in Werner syndrome cell lines.

Mutations in the chromosome 8p WRN gene cause Werner syndrome (WRN), a human autosomal recessive disease that mimics premature aging and is associated with genetic instability and an increased risk of cancer. All of the WRN mutations identified in WRN patients are predicted to truncate the WRN protein with loss of a C-terminal nuclear localization signal. However, many of these truncated proteins would retain WRN helicase and/or nuclease functional domains. We have used a combination of immune blot and immune precipitation assays to quantify WRN protein and its associated 3'-->5' helicase activity in genetically characterized WRN patient cell lines. None of the cell lines from patients harboring four different WRN mutations contained detectable WRN protein or immune-precipitable WRN helicase activity. Cell lines from WRN heterozygous individuals contained reduced amounts of both WRN protein and helicase activity. Quantitative immune blot analyses indicate that both lymphoblastoid cell lines and fibroblasts contain approximately 6 x 10(4)WRN molecules/cell. Our results indicate that most WRN mutations result in functionally equivalent null alleles, that WRN heterozygote effects may result from haploinsufficiency and that successful modeling of WRN pathogenesis in the mouse or in other model systems will require the use of WRN mutations that eliminate WRN protein expression.

Werner syndrome protein. I. DNA helicase and dna exonuclease reside on the same polypeptide.

Werner Syndrome (WS) is a human progeroid disorder characterized by genomic instability. The gene defective in WS encodes a 3' --> 5' DNA helicase (Gray, M. D., Shen, J.-C., Kamath-Loeb, A. S., Blank, A. , Sopher, B. L., Martin, G. M., Oshima, J., and Loeb, L. A.(1997) Nat. Genet. 17, 100-103). Sequence alignment analysis identified an N-terminal motif in WRN that is homologous to several exonucleases. Using combined molecular genetic, biochemical, and immunochemical approaches, we demonstrate that WRN also exhibits an integral DNA exonuclease activity. First, whereas wild-type recombinant WRN possesses both helicase and exonuclease activities, mutant WRN lacking the nuclease domain does not display exonucleolytic activity. In contrast, WRN proteins with defective helicase activity are active in exonucleolytic digestion of DNA. Second, the exonuclease co-purifies with the 160-kDa WRN protein and its associated DNA helicase and ATPase activities through successive steps of ion exchange and affinity chromatography, suggesting that all three activities are physically associated. Lastly, anti-WRN antiserum specifically co-precipitates the WRN helicase and exonuclease activities indicating that both activities reside on the same antigenic WRN polypeptide. The association of an exonuclease with WRN distinguishes it from other RecQ homologs and raises the possibility that the distinct phenotypic characteristics of WS may be due in part to a defective exonuclease.

Werner syndrome protein. II. Characterization of the integral 3' --> 5' DNA exonuclease.

In addition to its DNA helicase activity, Werner syndrome protein (WRN) also possesses an exonuclease activity (Shen, J.-C., Gray, M. D., Kamath-Loeb, A. S., Fry, M., Oshima, J., and Loeb, L. A. (1998) J. Biol. Chem. 273, 34139-34144). Here we describe the properties of nearly homogeneous WRN exonuclease. WRN exonuclease hydrolyzes a recessed strand in a partial DNA duplex but does not significantly digest single-stranded DNA, blunt-ended duplex, or a protruding strand of a partial duplex. Although DNA is hydrolyzed in the absence of nucleoside triphosphates, nuclease activity is markedly stimulated by ATP, dATP, or CTP. WRN exonuclease digests DNA with a 3' --> 5' directionality to generate 5'-dNMP products, and DNA strands terminating with either a 3'-OH or 3'-PO4 group are hydrolyzed to similar extents. A recessed DNA strand with a single 3'-terminal mismatch is hydrolyzed more efficiently by WRN than one with a complementary nucleotide, but the enzyme fails to hydrolyze a DNA strand terminating with two mismatched bases. WRN exonuclease is distinguished from known mammalian DNA nucleases by its covalent association with a DNA helicase, preference for a recessed DNA strand, stimulation by ATP, ability to equally digest DNA with 3'-OH or 3'-PO4 termini, and its preferential digestion of DNA with a single 3'-terminal mismatch.

The Werner syndrome protein is a DNA helicase.

Werner syndrome (WS) is an uncommon autosomal recessive disorder characterized by premature aging. The clinical manifestations of WS, including atherosclerosis and osteoporosis, appear early in adulthood, and death in the fourth to sixth decade commonly ensues from myocardial infarction or cancer. In accord with the aging phenotype, cells from WS patients have a reduced replicative life span in culture. Genomic instability is observed at the cytogenetic level in the form of chromosome breaks and translocations and at the molecular level by multiple large deletions. The Werner syndrome gene (WRN) has recently been cloned. The predicted product is a 1,432-amino-acid protein whose central domain is homologous to members of the RecQ family of DNA helicases. Such homology does not necessarily mean that WRN encodes an active helicase. For example, the Saccharomyces cerevisiae RAD26 gene protein and the human transcription-repair coupling factor CSB (Cockayne syndrome 8) are highly homologous to known helicases, yet neither encodes an active helicase. Moreover, the Bloom's syndrome gene (BLM), discovered before WRN, is also homologous to the RecQ family of DNA helicases, though we still await demonstration that it encodes an active helicase. Here we report that the WS protein does indeed catalyze DNA unwinding.

Mutagenesis by human immunodeficiency virus reverse transcriptase: incorporation of O6-methyldeoxyguanosine triphosphate.

The high frequency of incorporation of non-complementary nucleotides by HIV-1 reverse transcriptase is likely to be a major factor in the exceptionally rapid accumulation of viral mutations during the course of AIDS infections. To investigate whether this high level of infidelity is also associated with the incorporation of nucleotide analogs, we analyzed O6-methyldeoxyguanosine triphosphate and compared the incorporation of this analog by HIV-1 reverse transcriptase to that catalyzed by other DNA synthesizing enzymes. Our results indicate that O6-methyldeoxyguanosine triphosphate serves as a substrate for DNA synthesized in vitro by HIV-1 RT on both DNA and RNA templates. The product DNA contains the modified purine; it is sensitive to the repair enzyme, O6-methylguanine methyltransferase, which specifically reacts with DNA containing methylated guanines at the O6 position. Using a forward mutation assay we demonstrated that the nucleotide analog incorporated by HIV-1 RT is mutagenic. The mutations produced are single-base substitutions opposite template thymidines and result in A:T --> G:C transitions. The incorporation of a mutagenic nucleotide by HIV-1 RT highlights the possibility of increasing the rate of mutagenesis of HIV by the use of nucleotides that form non-complementary base pairs at high frequency.

Incorporation of the guanosine triphosphate analogs 8-oxo-dGTP and 8-NH2-dGTP by reverse transcriptases and mammalian DNA polymerases.

We have measured the efficiencies of utilization of 8-oxo-dGTP and 8-NH2-dGTP by human immunodeficiency virus type 1 and murine leukemia virus reverse transcriptases and compared them to those of DNA polymerases alpha and beta. Initially, we carried out primer extension reactions in the presence of dGTP or a dGTP analog and the remaining three dNTPs using synthetic DNA and RNA templates. These assays revealed that, in general, 8-NH2-dGTP is incorporated and extended more efficiently than 8-oxo-dGTP by all enzymes tested. Second, we determined rate constants for the incorporation of each analog opposite a template cytidine residue using steady state single nucleotide extension kinetics. Our results demonstrated the following. 1) Both reverse transcriptases incorporate the nucleotide analogs; discrimination against their incorporation is a function primarily of Km or Vmax depending on the analog and the enzyme. 2) Discrimination against the analogs is more stringent with the DNA template than with a homologous RNA template. 3) Polymerase alpha exhibits a mixed kinetic phenotype, with a large discrimination against 8-oxo-dGTP but a comparatively higher preference for 8-NH2-dGTP. 4) Polymerase beta incorporates both analogs efficiently; there is no discrimination with respect to Km and a significantly lower discrimination with respect to Vmax when compared with the other polymerases.

Inefficient repair of RNA x DNA hybrids.

RNA x DNA hybrids are commonly observed during normal biological processes. We tested the ability of three DNA-repair enzymes to remove lesions from the DNA strand of RNA x DNA heteroduplexes. Three nucleotide analogs, 5-hydroxy-2'-deoxycytidine triphosphate, 8-oxo-2'-deoxyguanosine triphosphate, and O6-methyl-2'-deoxyguanosine triphosphate, representative of lesions generated by oxygen damage and methylating agents, were incorporated into the DNA strand synthesized using either a DNA or RNA template. The extended DNA x DNA and RNA x DNA hybrids were used as substrates for bacterial formamidopyrimidine-DNA glycosylase, Nth protein (endonuclease III) and O6-methylguanine-DNA methyltransferase. We show that all three lesions are readily cleaved from the DNA strand of a DNA x DNA duplex but are relatively resistant to cleavage when present in the DNA strand of an RNA x DNA hybrid. Our in vitro studies suggest that damaged DNA in RNA x DNA hybrids is less likely to be repaired in vivo.

Analysis of three DnaK mutant proteins suggests that progression through the ATPase cycle requires conformational changes.

DnaK, the bacterial homolog of the eukaryotic hsp70 proteins, is an ATP-dependent chaperone whose basal ATPase is stimulated by synthetic peptides and its cohort heat shock proteins, DnaJ and GrpE. We have used three mutant DnaK proteins, E171K, D201N, and A174T (corresponding to Glu175, Asp206, and Ala179, respectively, in bovine heat stable cognate 70) to probe the ATPase cycle. All of the mutant proteins exhibit some alteration in basal ATP hydrolysis. However, they all exhibit more severe defects in the regulated activities. D201N and E171K are completely defective in all regulated activities of the protein and also in making the conformational change exhibited by the wt protein upon binding ATP. We suggest that the inability of D201N and E171K to achieve the ATP activated conformation prevents both stimulation by all effectors and the ATP-mediated release of GrpE. In contrast, the defect of A174T is much more specific. It exhibits normal binding and release of GrpE and normal stimulation of ATPase activity by DnaJ. However, it is defective in the synergistic activation of its ATPase by DnaJ and GrpE. We suggest that this mutant protein is specifically defective in a DnaJ/GrpE mediated conformational change in DnaK necessary for the synergistic action of DnaJ+GrpE.

Translational regulation of sigma 32 synthesis: requirement for an internal control element.

We have investigated the sequence requirements for the translational regulation of sigma 32 by examining the behavior of a new rpoH-lacZ protein fusion containing a short N-terminal fragment of sigma 32 fused to beta-galactosidase. Although the fusion retains rpoH translational initiation signals, it lacks translational regulation, implicating coding sequences within rpoH in this regulatory process.