DNA polymerase ι compensates for Fanconi anemia pathway deficiency by countering DNA replication stress

Fanconi anemia (FA) is caused by defects in cellular responses to DNA crosslinking damage and replication stress. Given the constant occurrence of endogenous DNA damage and replication fork stress, it is unclear why complete deletion of FA genes does not have a major impact on cell proliferation and germ-line FA patients are able to progress through development well into their adulthood. To identify potential cellular mechanisms that compensate for the FA deficiency, we performed dropout screens in FA mutant cells with a whole genome guide RNA library. This uncovered a comprehensive genome-wide profile of FA pathway synthetic lethality, including POLI and CDK4. As little is known of the cellular function of DNA polymerase iota (Pol ι), we focused on its role in the loss-of-function FA knockout mutants. Loss of both FA pathway function and Pol ι leads to synthetic defects in cell proliferation and cell survival, and an increase in DNA damage accumulation. Furthermore, FA-deficient cells depend on the function of Pol ι to resume replication upon replication fork stalling. Our results reveal a critical role for Pol ι in DNA repair and replication fork restart and suggest Pol ι as a target for therapeutic intervention in malignancies carrying an FA gene mutation.

Fanconi anemia (FA) is a genomic instability disorder caused by biallelic or x-linked mutations in any of 22 genes. FA patients are characterized by multiple developmental abnormalities, progressive bone marrow failure, and profound cancer susceptibility (1–3). Germ-line FA mutations predispose an individual to breast, ovarian, pancreatic, and hematological malignancies. Somatic FA mutations have been identified in sporadic acute leukemia and breast cancer (4–6).The FA pathway is the major cellular mechanism responding to DNA crosslinking damage and replication stress. The 22 FA gene products fall into several functional groups. In response to DNA damage, the FANCD2/FANCI complex is monoubiquitinated, signifying the activation of the canonical FA pathway (7, 8). The monoubiquitinated FANCD2/FANCI complex most likely orchestrates the recruitment of nucleolytic factors for the processing of crosslinking DNA damage (9, 10). The FA core complex, consisting of FANCA, -B, -C, -E, -F, -G, and -M, FAAP20, FAAP24, FAAP100, and the RING domain protein FANCL, provides the E3 ligase activity for the damage-induced monoubiquitination of FANCD2/FANCI (11–16). FANCP/XPF and FANCQ/SLX4, the third group of FA gene products, are nucleases or part of the nuclease scaffold, taking part in DNA cleavage for the removal of the crosslinking lesions (8, 17–21). DNA double-strand breaks, as an intermediate structure of ICL (Interstrand CrossLink) repair, depend on the fourth group of FA proteins, required in homologous recombination (FANCD1/BRCA2, FANCO/RAD51C, FANCJ/BARD1, and FANCR/RAD51) (22–26).In addition to the direct role in crosslinking damage repair, FA pathway components are linked to the protection of replication fork integrity during replication interruption that is not directly caused by damage to the DNA. BRCA1/2 are important in stabilizing stalled forks in an MRE11-dependent manner (27, 28). Similarly, FANCD2 and FANCI have been shown to prevent collapse of stalled replication forks (29, 30). Defects in the FA and recombination mechanisms lead to severe fork erosion and endogenous DNA damage accumulation upon reversible replication block, suggesting that the FA pathway plays a crucial role in DNA replication under both normal and perturbed growth conditions (8, 23, 31–34).Given the important role of the FA pathway in replication stress, it is perplexing that cells with a completely impaired FA mechanism are capable of sustained proliferation (34, 35). Overt abnormalities are absent in mice with knockout of several key FA genes (36–39). Moreover, individuals can survive without a functional FA pathway for decades (median life expectancy of 30 y for FA patients) (40). More recently, a genome-scale CRISPR-Cas9 guide RNA (gRNA) library screen has defined gene sets essential for proliferation of common model cell lines (41). None of the classic FA genes which participate in the monoubiquitination process appear to be essential in these screens. Cells deficient in classic FA genes can sustain growth despite the accumulation of endogenous DNA damage. Thus, it seems likely that compensatory mechanisms exist in FA mutant cells to support long-term viability.In this study, we sought to identify cellular mechanisms that are important for the survival of cells deficient in the FA pathway. Comparative genome-scale CRISPR/Cas9 screens were carried out in isogenic FA pathway-proficient and -deficient cells. Genes that exhibit synthetic lethality in FA mutant cells are candidates which compensate for the loss of the FA pathway function. Among the top candidates, we validated and investigated DNA polymerase (Pol) ι as a critical factor for the survival of FA mutant cells. We found that, in FA-deficient cells, Pol ι is crucial in the resumption of stressed replication forks and in suppressing the accumulation of endogenous DNA damage. This reveals a function for Pol ι in relieving DNA damage stress.     

A genetic system to identify DNA polymerase β mutator mutants

DNA polymerase β (pol β) is a 39-kDa protein that functions in DNA repair processes in mammalian cells. As a first step toward understanding mechanisms of polymerase fidelity, we developed a genetic method to identify mammalian pol β mutator mutants. This screen takes advantage of a microbial genetics assay and the ability of rat pol β to substitute for Escherichia coli DNA polymerase I in DNA replication in vivo. Using this screen, we identified 13 candidate pol β mutator mutants. Three of the candidate mutator mutants were further characterized in vivo and shown to confer an increased spontaneous mutation frequency over that of wild-type pol β to our bacterial strain. Purification and subsequent analysis of one of our putative mutator proteins, the pol β-14 protein, showed that it possesses intrinsic mutator activity in four different assays that measure the fidelity of DNA synthesis. Therefore, residue 265, which is altered in pol β-14 and another of our mutant proteins, pol β-166, is probably critical for accurate DNA synthesis by pol β. Thus, our genetic method of screening for pol β mutator mutants is useful in identifying active mammalian DNA polymerase mutants that encode enzymes that catalyze DNA synthesis with altered fidelity compared with the wild-type pol β enzyme.     

The β subunit sliding DNA clamp is responsible for unassisted mutagenic translesion replication by DNA polymerase III holoenzyme

The replication of damaged nucleotides that have escaped DNA repair leads to the formation of mutations caused by misincorporation opposite the lesion. In Escherichia coli, this process is under tight regulation of the SOS stress response and is carried out by DNA polymerase III in a process that involves also the RecA, UmuD′ and UmuC proteins. We have shown that DNA polymerase III holoenzyme is able to replicate, unassisted, through a synthetic abasic site in a gapped duplex plasmid. Here, we show that DNA polymerase III*, a subassembly of DNA polymerase III holoenzyme lacking the β subunit, is blocked very effectively by the synthetic abasic site in the same DNA substrate. Addition of the β subunit caused a dramatic increase of at least 28-fold in the ability of the polymerase to perform translesion replication, reaching 52% bypass in 5 min. When the ssDNA region in the gapped plasmid was extended from 22 nucleotides to 350 nucleotides, translesion replication still depended on the β subunit, but it was reduced by 80%. DNA sequence analysis of translesion replication products revealed mostly −1 frameshifts. This mutation type is changed to base substitution by the addition of UmuD′, UmuC, and RecA, as demonstrated in a reconstituted SOS translesion replication reaction. These results indicate that the β subunit sliding DNA clamp is the major determinant in the ability of DNA polymerase III holoenzyme to perform unassisted translesion replication and that this unassisted bypass produces primarily frameshifts.     

Interaction of human apurinic endonuclease and DNA polymerase β in the base excision repair pathway

Mutagenic abasic (AP) sites are generated directly by DNA-damaging agents or by DNA glycosylases acting in base excision repair. AP sites are corrected via incision by AP endonucleases, removal of deoxyribose 5-phosphate, repair synthesis, and ligation. Mammalian DNA polymerase β (Polβ) carries out most base excision repair synthesis and also can excise deoxyribose 5-phosphate after AP endonuclease incision. Yeast two-hybrid analysis now indicates protein–protein contact between Polβ and human AP endonuclease (Ape protein). In vitro, binding of Ape protein to uncleaved AP sites loads Polβ into a ternary complex with Ape and the AP-DNA. After incision by Ape, only Polβ exhibits stable DNA binding. Kinetic experiments indicated that Ape accelerates the excision of 5′-terminal deoxyribose 5-phosphate by Polβ. Thus, the two central players of the base excision repair pathway are coordinated in sequential reactions.     

Interlocking activities of DNA polymerase β in the base excision repair pathway

Base excision repair (BER) is a major cellular pathway for DNA damage repair. During BER, DNA polymerase β (Polβ) is hypothesized to first perform gap-filling DNA synthesis by its polymerase activity and then cleave a 5′-deoxyribose-5-phosphate (dRP) moiety via its dRP lyase activity. Through gel electrophoresis and kinetic analysis of partial BER reconstitution, we demonstrated that gap-filling DNA synthesis by the polymerase activity likely occurred after Schiff base formation but before β-elimination, the two chemical reactions catalyzed by the dRP lyase activity. The Schiff base formation and β-elimination intermediates were trapped by sodium borohydride reduction and identified by mass spectrometry and X-ray crystallography. Presteady-state kinetic analysis revealed that cross-linked Polβ (i.e., reduced Schiff base) exhibited a 17-fold higher polymerase efficiency than uncross-linked Polβ. Conventional and time-resolved X-ray crystallography of cross-linked Polβ visualized important intermediates for its dRP lyase and polymerase activities, leading to a modified chemical mechanism for the dRP lyase activity. The observed interlocking enzymatic activities of Polβ allow us to propose an altered mechanism for the BER pathway, at least under the conditions employed. Plausibly, the temporally coordinated activities at the two Polβ active sites may well be the reason why Polβ has both active sites embedded in a single polypeptide chain. This proposed pathway suggests a corrected facet of BER and DNA repair, and may enable alternative chemical strategies for therapeutic intervention, as Polβ dysfunction is a key element common to several disorders.

One of the major cellular pathways for repair of DNA damage is base excision repair (BER) (1–5). In this pathway (Scheme 1A), DNA lesions are removed by glycosylases (e.g., uracil by uracil–DNA glycosylase [UDG]) before the damaged DNA strand is incised by apurinic/apyrimidinic (AP) endonuclease, resulting in a single-nucleotide gap flanked by a 3′-OH and a 5′-deoxyribose-5-phosphate (dRP) moiety. Subsequently, DNA polymerase β (Polβ) is presumed to first catalyze gap-filling DNA synthesis through its DNA polymerase activity and then perform dRP cleavage via its dRP lyase activity, leaving a nicked DNA substrate for ligation by either Ligase III/XRCC1 or Ligase I (6–12).Open in a separate windowScheme 1.The BER pathway. (A) The BER pathway in the literature as cited in the Introduction. (B) Our proposed BER pathway.The dRP lyase active site resides within the 8-kDa N-terminal domain of Polβ, whereas the polymerase active site sits at the palm subdomain (SI Appendix, Fig. S1A) (7). Previously, Polβ has been shown to remove a dRP moiety through a Schiff base–mediated β-elimination reaction (13) rather than through hydrolysis (7–10, 14, 15). A Schiff base is generated following nucleophilic attack by the side chain of an active site lysine residue on the sugar C1′ atom of the dRP moiety (step 1 in Scheme 2). Whereas biochemical data suggest that K72 in human polymerase-β (hPolβ) acts as the active site nucleophile, conflicting evidence as well as a lack of supporting structural data have complicated understanding of the dRP cleavage mechanism (10, 14, 15). For instance, mutation of K72 to alanine does not fully abrogate the dRP lyase activity, suggesting that a different residue may support the nucleophilic attack on the C1′ (11). Furthermore, only limited conclusions can be drawn from the existing binary crystal structures of hPolβ bound to either a single-nucleotide gapped DNA substrate (hPolβ•DNA^P) (SI Appendix, Fig. S2B) containing only a 5′-phosphate, rather than a full dRP moiety, on the downstream primer (SI Appendix, Fig. S2 A, i) (16) or a nicked DNA substrate (hPolβ•DNA^THF) (SI Appendix, Fig. S2C) containing a 5′-dRP mimic (SI Appendix, Fig. S2 A, ii) (11). Due to the lack of the deoxyribose moiety in the structure of hPolβ•DNA^P, information about the dRP cleavage mechanism is lacking. On the other hand, in the structure of hPolβ•DNA^THF, the nonnatural dRP mimic was bound in a nonproductive docking site stabilized through the interaction between its 5′-phosphate and K68 (SI Appendix, Fig. S2C). This nonproductive site is distinct from the putative dRP lyase active site as the Nε atom of K72 is more than 10 Å from the dRP sugar C1′ (11). In fact, from this position, the dRP must rotate ∼120° around the 3′-phosphate to be in close-enough proximity to the Nε atom of K72 for nucleophilic attack to occur (SI Appendix, Fig. S2C). Furthermore, the active site residues responsible for stabilizing the reactive ring-opened aldehyde state of the dRP moiety and abstracting a proton from the ribose C2′ atom to facilitate β-elimination (Scheme 2) remain unidentified.Open in a separate windowScheme 2.Proposed chemical mechanism for the dRP lyase activity of hPolβ. Specific water molecules are denoted as X, Y, and Z.Biochemical studies of the processing of dRP moieties in yeast cell-free extract (17), steady-state kinetic studies of fully reconstituted human BER (4), and investigation of the numbers of endogenous AP sites in genomic DNA of rats and human tissue (5) all suggest that dRP cleavage is the rate-limiting step of the entire BER pathway. However, there is no experimental evidence to indicate that all potential steps associated with dRP cleavage by the lyase activity of Polβ (Scheme 2) occur after gap-filling DNA synthesis catalyzed by the polymerase activity. For example, if facile Schiff base formation occurs before and faster than nucleotide incorporation, the covalently linked Polβ–DNA intermediate, rather than the noncovalent binary complex Polβ•DNA, may catalyze gap-filling DNA synthesis. This possibility has never been investigated, and all previously published in vitro studies have used DNA substrates like either DNA^P (SI Appendix, Fig. S2 A, i) (18–24) or a gapped DNA substrate containing a dRP mimic (SI Appendix, Fig. S2 A, ii) (11, 25).Here, we generated a natural dRP moiety by using either UDG to process a nicked DNA substrate containing a 2′-deoxyuridine or UDG and apurinic/apyrimidinic endonuclease 1 (APE1) to initiate BER on a double-stranded DNA substrate containing a 2′-deoxyuridine. Addition of hPolβ, correct deoxynucleoside triphosphate (dNTP), and then, sodium borohydride (NaBH₄) to the dRP-containing DNA products allowed for the capture of a reduced Schiff base and a β-elimination intermediate produced via hPolβ-catalyzed dRP cleavage (Scheme 2). Through X-ray crystallographic, kinetic, and mass spectrometric (MS) analysis of these cross-linked hPolβ complexes, we envisioned a detailed chemical mechanism for the dRP lyase activity of hPolβ. In addition, we utilized presteady-state kinetic methods to evaluate the impact of the reduced Schiff base intermediate on the efficiency and fidelity of gap-filling DNA synthesis by the polymerase activity of hPolβ. Finally, we employed time-resolved X-ray crystallography to structurally characterize intermediates of gap-filling DNA synthesis by cross-linked hPolβ. Based on several lines of experimental evidence, we proposed a modified BER pathway (Scheme 1B), which posits an interlocking mechanism in which gap-filling DNA synthesis by the polymerase activity occurs between Schiff base formation and β-elimination, the two steps catalyzed by the lyase activity.     

DNA polymerase δ isolated from Schizosaccharomyces pombe contains five subunits

DNA polymerase δ (pol δ) plays an essential role in DNA replication, repair, and recombination. We have purified pol δ from Schizosaccharomyces pombe more than 10³-fold and demonstrated that the polymerase activity of purified S. pombe pol δ is completely dependent on proliferating cell nuclear antigen and replication factor C. SDS/PAGE analysis of the purified fraction indicated that the pol δ complex consists of five subunits that migrate with apparent molecular masses of 125, 55, 54, 42, and 22 kDa. Western blot analysis indicated that the 125, 55, and 54 kDa proteins are the large catalytic subunit (Pol3), Cdc1, and Cdc27, respectively. The identity of the other two subunits, p42 and p22, was determined following proteolytic digestion and sequence analysis of the resulting peptides. The peptide sequences derived from the p22 subunit indicated that this subunit is identical to Cdm1, previously identified as a multicopy suppressor of the temperature-sensitive cdc1-P13 mutant, whereas peptide sequences derived from the p42 subunit were identical to a previously uncharacterized ORF located on S. pombe chromosome 1.     

An enhancer-blocking element between α and δ gene segments within the human T cell receptor α/δ locus

T cell receptor (TCR) α and δ gene segments are organized within a single genetic locus but are differentially regulated during T cell development. An enhancer-blocking element (BEAD-1, for blocking element alpha/delta 1) was localized to a 2.0-kb region 3′ of TCR δ gene segments and 5′ of TCR α joining gene segments within this locus. BEAD-1 blocked the ability of the TCR δ enhancer (E_δ) to activate a promoter when located between the two in a chromatin-integrated construct. We propose that BEAD-1 functions as a boundary that separates the TCR α/δ locus into distinct regulatory domains controlled by E_δ and the TCR α enhancer, and that it prevents E_δ from opening the chromatin of the TCR α joining gene segments for VDJ recombination at an early stage of T cell development.     

Molecular analysis of mutations in DNA polymerase η in xeroderma pigmentosum-variant patients

Xeroderma pigmentosum variant (XP-V) cells are deficient in their ability to synthesize intact daughter DNA strands after UV irradiation. This deficiency results from mutations in the gene encoding DNA polymerase eta, which is required for effecting translesion synthesis (TLS) past UV photoproducts. We have developed a simple cellular procedure to identify XP-V cell strains, and have subsequently analyzed the mutations in 21 patients with XP-V. The 16 mutations that we have identified fall into three categories. Many of them result in severe truncations of the protein and are effectively null alleles. However, we have also identified five missense mutations located in the conserved catalytic domain of the protein. Extracts of cells falling into these two categories are defective in the ability to carry out TLS past sites of DNA damage. Three mutations cause truncations at the C terminus such that the catalytic domains are intact, and extracts from these cells are able to carry out TLS. From our previous work, however, we anticipate that protein in these cells will not be localized in the nucleus nor will it be relocalized into replication foci during DNA replication. The spectrum of both missense and truncating mutations is markedly skewed toward the N-terminal half of the protein. Two of the missense mutations are predicted to affect the interaction with DNA, the others are likely to disrupt the three-dimensional structure of the protein. There is a wide variability in clinical features among patients, which is not obviously related to the site or type of mutation.     

Evidence for a role for DNA polymerase β in mammalian meiosis

DNA polymerase β (pol β) is an enzyme possessing both polymerase and deoxyribose phophatase activities. Although pol β is not believed to participate in the replication of genomic DNA, several studies have indicated a role for pol β in DNA repair. The high level of expression of pol β in mouse and rat testes raises the possibility that pol β participates in mammalian meiosis. Using antibody localization, we detect foci that stain with pol β antisera at discrete sites along homologous chromosomes as they synapse and progress through prophase of meiosis I. These data suggest that pol β participates in meiotic events associated with synapsis and recombination.     

Global genomic instability caused by reduced expression of DNA polymerase ε in yeast

DNA polymerase ε (Pol ε) is one of the three replicative eukaryotic DNA polymerases. Pol ε deficiency leads to genomic instability and multiple human diseases. Here, we explored global genomic alterations in yeast strains with reduced expression of POL2, the gene that encodes the catalytic subunit of Pol ε. Using whole-genome SNP microarray and sequencing, we found that low levels of Pol ε elevated the rates of mitotic recombination and chromosomal aneuploidy by two orders of magnitude. Strikingly, low levels of Pol ε resulted in a contraction of the number of repeats in the ribosomal DNA cluster and reduced the length of telomeres. These strains also had an elevated frequency of break-induced replication, resulting in terminal loss of heterozygosity. In addition, low levels of Pol ε increased the rate of single-base mutations by 13-fold by a Pol ζ-dependent pathway. Finally, the patterns of genomic alterations caused by low levels of Pol ε were different from those observed in strains with low levels of the other replicative DNA polymerases, Pol α and Pol δ, providing further insights into the different roles of the B-family DNA polymerases in maintaining genomic stability.

Chromosome replication in eukaryotes requires three conserved B-family polymerases (Pols α, δ, and ε), with DNA polymerase ζ having an important role in the replication of damaged bases (1, 2). The roles of these polymerases in duplicating the genome are different. Polymerase (Pol) α initiates DNA replication by synthesizing short RNA/DNA primers, and Pol δ is required to extend these fragments to complete lagging-strand replication. Although it is widely accepted that the main role of Pol ε is to replicate the leading-strand template, more recent evidence suggests that in conditions of replication stress, Pol δ can also participate in leading-strand replication (3, 4). Although the catalytic domain of Pol ε is not required for viability in yeast (5), Pol ε is required for assembly of the replisome (6). Mutations of POL2 (encoding the catalytic subunit of Pol ε) also result in defects in S-phase checkpoint activation (7), short telomeres (8), and modest defects in DNA repair (9). In addition, mutations of DPB3 (encoding a nonessential subunit of Pol ε) lead to a reduction in the efficiency of silencing of markers inserted in the ribosomal DNA (rDNA) (10).In humans, mutations in Pol ε result in several human immunodeficiency syndromes (11, 12). In addition, mutations in POLE (equivalent to the POL2 gene in yeast) are associated with a variety of types of tumors with a mutator phenotype (13, 14). The cancer-associated mutations are found throughout the gene but concentrated within the exonuclease domain. Tumors with elevated mutation rates are often heterozygous for the POLE mutation (13), suggesting the possibility that a reduction in the amount of the wild-type enzyme might be relevant to the mutator phenotype. In this study, we examine the effects of reducing the amount of Pol ε on a variety of genomic alterations, including single-base substitutions, insertions/deletions (in/dels), changes in chromosome structure, and alterations in chromosome number.Previously, we showed that reduced expression of POL1 (encoding the catalytic subunit of Pol α) or POL3 (encoding the catalytic subunit of Pol δ) in Saccharomyces cerevisiae greatly elevated aneuploidy, chromosome rearrangements, and loss of heterozygosity (LOH) (15–18). Our analysis suggested that most of these events reflected double-strand DNA breaks (DSBs) that occurred at stalled or broken replication forks. Although reduced levels of both Pol α and Pol δ greatly elevated the rates of LOH, and large deletions and duplications (often caused by homologous recombination between nonallelic Ty retrotransposons), there were a number of differences in the genomic alterations induced by these two polymerases (19). For example, the ratio of LOH events to chromosome losses is about fourfold higher in strains with low levels of Pol α than in strains with low Pol δ. The likely explanation of this ratio difference is that Pol δ has a more important role in the repair of DSBs by homologous recombination than Pol α. Thus, many DSBs in strains with low Pol δ are not repaired, leading to chromosome loss (19).In the present study, we explored the global genomic instability in yeast strains in which POL2, encoding the catalytic subunit of Pol ε, was down-regulated. Using a combination of single-nucleotide polymorphism (SNP) microarrays and whole-genome sequencing, we found that these strains have substantially elevated LOH events, chromosome aneuploidy, and single-base substitutions. We found that low Pol ε induced a different pattern of genomic instability than induced by low levels of Pol α or Pol δ, providing insights into how B-family DNA polymerases coordinately contribute to genomic stability in eukaryotic cells.     

Reciprocal regulation of Gsα by palmitate and the βγ subunit

Hormonal activation of G_s, the stimulatory regulator of adenylyl cyclase, promotes dissociation of α_s from Gβγ, accelerates removal of covalently attached palmitate from the Gα subunit, and triggers release of a fraction of α_s from the plasma membrane into the cytosol. To elucidate relations among these three events, we assessed biochemical effects in vitro of attached palmitate on recombinant α_s prepared from Sf9 cells. In comparison to the unpalmitoylated protein (obtained from cytosol of Sf9 cells, treated with a palmitoyl esterase, or expressed as a mutant protein lacking the site for palmitoylation), palmitoylated α_s (from Sf9 membranes, 50% palmitoylated) was more hydrophobic, as indicated by partitioning into TX-114, and bound βγ with 5-fold higher affinity. βγ protected GDP-bound α_s, but not α_s· GTP[γS], from depalmitoylation by a recombinant esterase. We conclude that βγ binding and palmitoylation reciprocally potentiate each other in promoting membrane attachment of α_s and that dissociation of α_s·GTP from βγ is likely to mediate receptor-induced α_s depalmitoylation and translocation of the protein to cytosol in intact cells.     

Inhibition of the electrostatic interaction between β-amyloid peptide and membranes prevents β-amyloid-induced toxicity

The accumulation of β-amyloid peptides (Aβ) into senile plaques is one of the hallmarks of Alzheimer disease. Aggregated Aβ is toxic to cells in culture and this has been considered to be the cause of neurodegeneration that occurs in the Alzheimer disease brain. The discovery of compounds that prevent Aβ toxicity may lead to a better understanding of the processes involved and ultimately to possible therapeutic drugs. Low nanomolar concentrations of Aβ1-42 and the toxic fragment Aβ25-35 have been demonstrated to render cells more sensitive to subsequent insults as manifested by an increased sensitivity to formazan crystals following MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reduction. Formation of the toxic β-sheet conformation by Aβ peptides is increased by negatively charged membranes. Here we demonstrate that phloretin and exifone, dipolar compounds that decrease the effective negative charge of membranes, prevent association of Aβ1-40 and Aβ25-35 to negatively charged lipid vesicles and Aβ induced cell toxicity. These results suggest that Aβ toxicity is mediated through a nonspecific physicochemical interaction with cell membranes.     

The enzymological basis for resistance of herpesvirus DNA polymerase mutants to acyclovir: Relationship to the structure of α-like DNA polymerases

Acyclovir (ACV), like many antiviral drugs, is a nucleoside analog. In vitro, ACV triphosphate inhibits herpesvirus DNA polymerase by means of binding, incorporation into primer/template, and dead-end complex formation in the presence of the next deoxynucleoside triphosphate. However, it is not known whether this mechanism operates in vivo. To address this and other questions, we analyzed eight mutant polymerases encoded by drug-resistant viruses, each altered in a region conserved among α-like DNA polymerases. We measured K_m and k_cat values for dGTP and ACV triphosphate incorporation and K_i values of ACV triphosphate for dGTP incorporation for each mutant. Certain mutants showed increased K_m values for ACV triphosphate incorporation, suggesting a defect in inhibitor binding. Other mutants showed reduced k_cat values for ACV triphosphate incorporation, suggesting a defect in incorporation of inhibitor into DNA, while the rest of the mutants exhibited both altered k_m and k_cat values. In most cases, the fold increase in K_i of ACV triphosphate for dGTP incorporation relative to wild-type polymerase was similar to fold resistance conferred by the mutation in vivo; however, one mutation conferred a much greater increase in resistance than in K_i. The effects of mutations on enzyme kinetics could be explained by using a model of an α-like DNA polymerase active site bound to primer/template and inhibitor. The results have implications for mechanisms of action and resistance of antiviral nucleoside analogs in vivo, in particular for the importance of incorporation into DNA and for the functional roles of conserved regions of polymerases.     

Novel α + β Zr Alloys with Enhanced Strength

Low-alloyed zirconium alloys are widely used in nuclear applications due to their low neutron absorption cross-section. These alloys, however, suffer from limited strength. Well-established guidelines for the development of Ti alloys were applied to design new two-phase ternary Zr alloys with improved mechanical properties. Zr-4Sn-4Nb and Zr-8Sn-4Nb alloys have been manufactured by vacuum arc melting, thermo-mechanically processed by annealing, forging, and aging to various microstructural conditions and thoroughly characterized. Detailed Scanning electron microscopy (SEM) analysis showed that the microstructural response of the alloys is rather similar to alpha + beta Ti alloys. Duplex microstructure containing primary alpha phase particles surrounded by lamellar alpha + beta microstructure can be achieved by thermal processing. Mechanical properties strongly depend on the previous treatment. Ultimate tensile strength exceeding 700 MPa was achieved exceeding the strength of commercial Zr alloys for nuclear applications by more than 50%. Such an improvement in strength more than compensates for the increased neutron absorption cross-section. This study aims to exploit the potential of alpha + beta Zr alloys for nuclear applications.     

Integrin αvβ1 promotes infection by human metapneumovirus

Human metapneumovirus (hMPV) is a recently described paramyxovirus that causes lower respiratory infections in children and adults worldwide. The hMPV fusion (F) protein is a membrane-anchored glycoprotein and major protective antigen. All hMPV F protein sequences determined to date contain an Arg-Gly-Asp (RGD) sequence, suggesting that F engages RGD-binding integrins to mediate cell entry. The divalent cation chelator EDTA, which disrupts heterodimeric integrin interactions, inhibits infectivity of hMPV but not the closely related respiratory syncytial virus (RSV), which lacks an RGD motif. Function-blocking antibodies specific for αvβ1 integrin inhibit infectivity of hMPV but not RSV. Transfection of nonpermissive cells with αv or β1 cDNAs confers hMPV infectivity, whereas reduction of αv and β1 integrin expression by siRNA inhibits hMPV infection. Recombinant hMPV F protein binds to cells, whereas Arg-Gly-Glu (RGE)-mutant F protein does not. These data suggest that αvβ1 integrin is a functional receptor for hMPV.     

Noncooperativity of the αβ Dimer in the Reaction of Hemoglobin with Oxygen

The theory that the alphabeta dimer is the functional unit of cooperativity in hemoglobin has been tested by determination of the oxygen equilibrium curve of stable deoxy dimers, obtained by the addition of 0.9 M MgCl(2) to human des-Arg 141alpha-hemoglobin. Cooperativity was absent in this medium, but was regained on transfer of the hemoglobin to a dilute phosphate buffer, where tetramers reformed. X-ray analysis of crystals of oxy- and deoxy-des-Arg hemoglobins showed that the removal of Arg 141alpha would leave the structure of alphabeta dimers unchanged. Nonreactivity of the sulfhydryl groups at 112beta proved that the subunits in deoxy dimers form the same contact as in oxy dimers, namely alpha(1)beta(1), and that no significant dissociation into free subunits occurs in 0.9 M MgCl(2). The absorption spectrum of the deoxy dimers corresponded to the sum of the spectra of the free deoxy alpha and beta subunits, and was different from that of the deoxy tetramer, showing the constraining salt bridges formed by the C-terminal residues in the tetramer to be necessary for the spectral changes normally observed on association of the deoxy subunits.