Determinants of DNA mismatch recognition within the polymerase domain of the Klenow fragment.

The Klenow fragment of Escherichia coli DNA polymerase I catalyzes template-directed synthesis of DNA and uses a separate 3'-5' exonuclease activity to edit misincorporated bases. The polymerase and exonuclease activities are contained in separate structural domains. In this study, nine Klenow fragment derivatives containing mutations within the polymerase domain were examined for their interaction with model primer-template duplexes. The partitioning of the DNA primer terminus between the polymerase and 3'-5' exonuclease active sites of the mutant proteins was assessed by time-resolved fluorescence anisotropy, utilizing a dansyl fluorophore attached to the DNA. Mutation of N845 or R668 disrupted favorable interactions between the Klenow fragment and a duplex containing a matched terminal base pair but had little effect when the terminus was mismatched. Thus, N845 and R668 are required for recognition of correct terminal base pairs in the DNA substrate. Mutation of N675, R835, R836, or R841 resulted in tighter polymerase site binding of DNA, suggesting that the side chains of these residues induce strain in the DNA and/or protein backbone. A double mutant (N675A/R841A) showed an even greater polymerase site partitioning than was displayed by either single mutation, indicating that such strain is additive. In both groups of mutant proteins, the ability to discriminate between duplexes containing matched or mismatched base pairs was impaired. In contrast, mutation of K758 or Q849 had no effect on partitioning relative to wild type, regardless of DNA mismatch character. These results demonstrate that DNA mismatch recognition is dependent on specific amino acid residues within the polymerase domain and is not governed solely by thermodynamic differences between correct and mismatched base pairs. Moreover, this study suggests a mechanism whereby the Klenow fragment is able to recognize polymerase errors following a misincorporation event, leading to their eventual removal by the 3'-5' exonuclease activity.     

Increased risk of lung cancer associated with a functionally impaired polymorphic variant of the human DNA glycosylase NEIL2

Human NEIL2, one of five oxidized base-specific DNA glycosylases, is unique in preferentially repairing oxidative damage in transcribed genes. Here we show that depletion of NEIL2 causes a 6-7-fold increase in spontaneous mutation frequency in the HPRT gene of the V79 Chinese hamster lung cell line. This prompted us to screen for NEIL2 variants in lung cancer patients' genomic DNA. We identified several polymorphic variants, among which R103Q and R257L were frequently observed in lung cancer patients. We then characterized these variants biochemically, and observed a modest decrease in DNA glycosylase activity relative to the wild type (WT) only with the R257L mutant protein. However, in reconstituted repair assays containing WT NEIL2 or its R257L and R103Q variants together with other DNA base excision repair (BER) proteins (PNKP, Polβ, Lig IIIα and XRCC1) or using NEIL2-FLAG immunocomplexes, an ~5-fold decrease in repair was observed with the R257L variant compared to WT or R103Q NEIL2, apparently due to the R257L mutant's lower affinity for other repair proteins, particularly Polβ. Notably, increased endogenous DNA damage was observed in NEIL2 variant (R257L)-expressing cells relative to WT cells. Taken together, our results suggest that the decreased DNA repair capacity of the R257L variant can induce mutations that lead to lung cancer development.     

Characterization of the sequence-specific interaction of mouse c-myb protein with DNA.

We have examined parameters that affect sequence-specific interactions of the mouse c-myb protein with DNA oligomers containing the Myb-binding motif (CA/CGTTPu). Complexes formed between these oligomers and in vitro translated c-myb proteins were analysed by electrophoresis on non-denaturing polyacrylamide gels using the mobility-shift assay. By progressive truncation of c-myb coding sequences it was demonstrated that amino acids downstream of a region of three imperfect 51-52 residue repeats (designated R1, R2 and R3), which are located close to the amino terminus of the protein, had no qualitative or quantitative effect on the ability to interact specifically with this DNA motif. However, removal of only five amino acids of the R3 repeat completely abolished this activity. The contribution of individual DNA-binding domain repeats to this interaction was investigated by precisely deleting each individually: it was demonstrated that a combination of R2 and R3 was absolutely required for complex formation while the R1 repeat was completely dispensible. c-myb proteins showed quantitatively greater interaction with oligomers containing duplicated rather than single Myb-binding motif, in particular where these were arranged in tandem. Moreover, it was observed that c-myb protein interacted with these tandem motifs as a monomer. These findings imply that a single protein subunit straddles adjacent binding sites and the implications for c-myb activity are discussed.     

Reduced Nuclease Activity of Apurinic/Apyrimidinic Endonuclease (APE1) Variants on Nucleosomes: IDENTIFICATION OF ACCESS RESIDUES*

Non-coding apurinic/apyrimidinic (AP) sites are generated at high frequency in genomic DNA via spontaneous hydrolytic, damage-induced or enzyme-mediated base release. AP endonuclease 1 (APE1) is the predominant mammalian enzyme responsible for initiating removal of mutagenic and cytotoxic abasic lesions as part of the base excision repair (BER) pathway. We have examined here the ability of wild-type (WT) and a collection of variant/mutant APE1 proteins to cleave at an AP site within a nucleosome core particle. Our studies indicate that, in comparison to the WT protein and other variant/mutant enzymes, the incision activity of the tumor-associated variant R237C and the rare population variant G241R are uniquely hypersensitive to nucleosome complexes in the vicinity of the AP site. This defect appears to stem from an abnormal interaction of R237C and G241R with abasic DNA substrates, but is not simply due to a DNA binding defect, as the site-specific APE1 mutant Y128A, which displays markedly reduced AP-DNA complex stability, did not exhibit a similar hypersensitivity to nucleosome structures. Notably, this incision defect of R237C and G241R was observed on a pre-assembled DNA glycosylase·AP-DNA complex as well. Our results suggest that the BER enzyme, APE1, has acquired distinct surface residues that permit efficient processing of AP sites within the context of protein-DNA complexes independent of classic chromatin remodeling mechanisms.     

Biochemical characterization of a SET and transposase fusion protein, Metnase: its DNA binding and DNA cleavage activity

Metnase (SETMAR) is a SET and transposase fusion protein that promotes in vivo end joining activity and mediates genomic integration of foreign DNA. Recent studies showed that Metnase retained most of the transposase activities, including 5'-terminal inverted repeat (TIR)-specific binding and assembly of a paired end complex, and cleavage of the 5'-end of the TIR element. Here we show that R432 within the helix-turn-helix motif is critical for sequence-specific recognition, as the R432A mutation abolishes its TIR-specific DNA binding activity. Metnase possesses a unique DNA nicking and/or endonuclease activity that mediates cleavage of duplex DNA in the absence of the TIR sequence. While the HTH motif is essential for the Metnase-TIR interaction, it is not required for its DNA cleavage activity. The DDE-like motif is crucial for its DNA cleavage action as a point mutation at this motif (D483A) abolished its DNA cleavage activity. Together, our results suggest that Metnase's DNA cleavage activity, unlike those of other eukaryotic transposases, is not coupled to its sequence-specific DNA binding.     

Cloning and mutagenesis of the Rhizobium meliloti isocitrate dehydrogenase gene.

The gene encoding Rhizobium meliloti isocitrate dehydrogenase (ICD) was cloned by complementation of an Escherichia coli icd mutant with an R. meliloti genomic library constructed in pUC18. The complementing DNA was located on a 4.4-kb BamHI fragment. It encoded an ICD that had the same mobility as R. meliloti ICD in nondenaturing polyacrylamide gels. In Western immunoblot analysis, antibodies raised against this protein reacted with R. meliloti ICD but not with E. coli ICD. The complementing DNA fragment was mutated with transposon Tn5 and then exchanged for the wild-type allele by recombination by a novel method that employed the Bacillus subtilis levansucrase gene. No ICD activity was found in the two R. meliloti icd::Tn5 mutants isolated, and the mutants were also found to be glutamate auxotrophs. The mutants formed nodules, but they were completely ineffective. Faster-growing pseudorevertants were isolated from cultures of both R. meliloti icd::Tn5 mutants. In addition to lacking all ICD activity, the pseudorevertants also lacked citrate synthase activity. Nodule formation by these mutants was severely affected, and inoculated plants had only callus structures or small spherical structures.     

Expression of acetylcholinesterase gene(s) in the human brain: molecular cloning evidence for cross-homologous sequences

The regulation of acetylcholinesterase (AChE) in the human brain has been approached at the level of the genome. A human DNA fragment of the length of 2 600 nucleotides was isolated from a human genomic library. This DNA fragment, designated Huache 1R, bears sequence homology to a DNA fragment from the vicinity of the Drosophila Ace region, that controls AChE biosynthesis (Soreq et al., 1985). Polyadenylated RNA from human brain was hybridized with Huache 1R DNA, eluted and microinjected into Xenopus oocytes in the absence or presence of 35S-methionine. The hybrid-selected RNA induced the biosynthesis of active AChE in the oocytes. Immunoprecipitation of labeled oocyte proteins with monoclonal antibodies against human AChE (Fambrough et al., 1982) resulted in the selective precipitation of an 85 000 Mr induced protein, with a similar size to that of the subunit of human brain AChE. These findings show that the Huache 1R DNA hybridizes with human brain AChEmRNA. The Huache 1R fragment was employed to select a collection of 12 homologous phage-cloned human genomic DNA fragments with different restriction patterns. A cDNA library in pBR322 plasmids was prepared from polyadenylated RNA isolated from embryonic brain. This library was also screened using labeled Huache 1R DNA as a probe. Forty-two out of 37 000 colonies were found positive. Several of these were selected for further analyses. Hybrid-selection experiments using DNA from two of the positive plasmid clones showed that these cDNAs also hybridize with AChEmRNA from human brain. DNA blot hybridization revealed homologies between these cDNA chains and the original Huache 1 fragment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of sodium butyrate and 5-azacytidine on DNA methylation in human tumor cell lines: variable response to drug treatment and withdrawal.

The effect of butyrate (Btr) and 5-azacytidine (azaC) on genomic DNA methylation was examined in a variety of human tumor cell lines. Butyrate treatment differentially affected 5-methylcytosine (m5C) content in a cell type-specific manner. For example, incubation of CBT cells (glioblastoma multiforme) for 48 h in medium containing 2 mM Btr resulted in the hypomethylation of their DNA; after removal of the drug, the m5C content was rapidly (24 h) restored to its original level. In contrast, when HeLa cells (cervical carcinoma) were cultured for 48 h in 3 mM Btr, their genomic DNA became more extensively methylated relative to the untreated control, and the hypermethylated state was maintained for long periods in culture. Treatment of HeLa cell lines with azaC resulted in extensive demethylation of their DNA. Following removal of the drug, the DNA was rapidly remethylated to levels equal to or greater than those of the untreated cells. The hypermethylated state has remained stable for over 180 generations. Together, the data suggest that cells respond rapidly to alter drug-induced reductions in the m5C content of their DNA, and this response can subsequently result in overmethylation. In contrast, when drug-induced hypermethylation of genomic DNA occurs, it may persist for many cell generations.     

Characterization of RR-1 and RR-2 cuticular proteins from Myzus persicae

A cDNA library for Myzus persicae has served to identify sequences coding for cuticular proteins (CPs) with RR-1 and RR-2 consensus. Two putative CPs showed a common RR-2 chitin binding domain (CBD) but differed in their C and N terminals. Two other predicted CPs showed a typical RR-1 CBD but differed in size and sequence of the C and N terminals. An additional sequence encoding for a protein that showed terminal amino acid repeats similar to those of putative CPs from M. persicae, but lacked the R & R consensus, was also described. A comparison of the sequences obtained from the cDNA library with those attained from the genomic DNA, confirmed their identity as cuticular proteins genes. Presence of introns was revealed in the Mpcp4 and Mpcp5 genes coding for CPs with an RR-1 consensus. The Mpcp4 has a single large intron, while the Mpcp5 has two shorter ones. Introns were not found in the Mpcp2 and Mpcp3 genes encoding for CPs with RR-2 consensus. Differences were also noticed for 3' UTR and 5' UTR of both the RR-1 and RR-2 CPs. CPs genes were expressed in bacteria, and the resulting protein was identified as a CP by amino acid sequencing.     

Identification of a DNA processing complex from Deinococcus radiodurans

An efficient DNA strand break repair contributes to the radioresistance of Deinococcus radiodurans, which harbors the DNA repair pathways nearly identical to Escherichia coli. The molecular mechanisms of these proteins functioning in 2 diverse classes of bacteria seem to be different. The macromolecular interactions and formation of multiprotein complexes in vivo have gained significant importance in explaining the mechanism of the complex cellular processes. Here, we report the identification of a novel DNA metabolic protein complex from D. radiodurans. A similar complex has, however, not been found in E. coli. Mass spectrometric analysis showed the presence of a few known DNA repair proteins, molecular chaperones, and a large number of uncharacterized proteins from D. radiodurans R1. Biochemical and immunoblotting results indicated the presence of the protein promoting DNA repair A, DNA polymerase, Mg2+, and (or) Mn2+ -dependent 5'-->3' exonuclease activity along with protein kinase activity and phosphoproteins. DNA ligase activity was completely dependent upon the ATP requirement, as no ligase activity was seen in the presence of NAD as a cofactor. These results suggest the molecular interactions of the known DNA repair proteins with uncharacterized proteins in the macromolecular complex and the regulation of DNA degradation with the involvement of ATP and protein kinase functions.     

Murine leukemia virus R Peptide inhibits influenza virus hemagglutinin-induced membrane fusion

The cytoplasmic tail of the murine leukemia virus (MuLV) envelope (Env) protein is known to play an important role in regulating viral fusion activity. Upon removal of the C-terminal 16 amino acids, designated as the R peptide, the fusion activity of the Env protein is activated. To extend our understanding of the inhibitory effect of the R peptide and investigate the specificity of inhibition, we constructed chimeric influenza virus-MuLV hemagglutinin (HA) genes. The influenza virus HA protein is the best-studied membrane fusion model, and we investigated the fusion activities of the chimeric HA proteins. We compared constructs in which the coding sequence for the cytoplasmic tail of the influenza virus HA protein was replaced by that of the wild-type or mutant MuLV Env protein or in which the cytoplasmic tail sequence of the MuLV Env protein was added to the HA cytoplasmic domain. Enzyme-linked immunosorbent assays and Western blot analysis showed that all chimeric HA proteins were effectively expressed on the cell surface and cleaved by trypsin. In BHK21 cells, the wild-type HA protein had a significant ability after trypsin cleavage to induce syncytium formation at pH 5.1; however, neither the chimeric HA protein with the full-length cytoplasmic tail of MuLV Env nor the full-length HA protein followed by the R peptide showed any syncytium formation. When the R peptide was truncated or mutated, the fusion activity was partially recovered in the chimeric HA proteins. A low-pH conformational-change assay showed that similar conformational changes occurred for the wild-type and chimeric HA proteins. All chimeric HA proteins were capable of promoting hemifusion and small fusion pore formation, as shown by a dye redistribution assay. These results indicate that the R peptide of the MuLV Env protein has a sequence-dependent inhibitory effect on influenza virus HA protein-induced membrane fusion and that the inhibitory effect occurs at a late stage in fusion pore enlargement.     

Use of conversion adaptors to clone antigen genes in lambda gt11

A strategy has been devised and tested to employ EcoRI conversion adaptors for cloning 5' cohesive-ended restriction fragments into the unique EcoRI site of the lambda gt11 expression vector. Five lambda gt11 chromosomal libraries were constructed with Rickettsia tsutsugamushi genomic DNA digested with restriction enzymes generating five different 5' cohesive ends. Recombinant phage yields as high as 10(7) plaque forming units were achieved without amplification of the five libraries. Sequences encoding epitopes of all eight R. tsutsugamushi polypeptide antigens, previously identified by Western blot analysis, were obtained in the five lambda gt11 expression libraries. Recombinant antigen expression was dependent on lambda gt11 lac promoter induction in 39% of the recombinants assayed. This method significantly improves the efficiency of genomic lambda gt11 library construction by eliminating blunt-ended ligation and simplifying the removal of unligated EcoRI-ended oligonucleotides.     

A novel route for immobilization of proteins to silica particles incorporating silaffin domains

In the diatom Cylindrotheca fusiformis, modified peptides called silaffin polypeptides are responsible for silica deposition in vivo at ambient conditions. Recently, it was discovered that the synthetic R5 peptide, the repeat unit of silaffin polypeptide without post‐translational modification, was capable of precipitating silica in vitro and at ambient conditions. Herein, chimeric proteins were generated by incorporating synthetic silaffin R5 peptides and related unmodified silaffin domains (R1–R7) from Cylindrotheca fusiformis onto green fluorescent protein (GFP) by recombinant DNA technology and their ability to cause silicification was also examined. GFP chimeric proteins showed silicification at very low concentrations (600–700 μg/mL) when compared with adding excess amounts of R5 peptides (10 mg/mL) as previously reported. Sensitive to pH conditions, only the GFP‐R1 chimera showed silicification activity at pH 8.0. The protein immobilization efficiencies of these chimeras were unexpectedly high ranging from 75 to 85%, with the R1 silaffin‐protein construct showing excellent immobilization efficiency and a constant molar ratio of silica to protein ranging from 250 to 350 over a wide pH range. The average silica particle sizes had a tendency to decrease as pH increased to basic conditions. This study demonstrated the production of nanoscale immobilized protein, fabricated via silaffin‐fused proteins. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Construction of TnphoA gene fusions in Rhodobacter sphaeroides: isolation and characterization of a respiratory mutant unable to utilize dimethyl sulfoxide as a terminal electron acceptor during anaerobic growth in the dark on glucose.

We have constructed a suicide vector, pU1800, containing the transposable element TnphoA (Tn5 IS50L::phoA), for the purpose of producing protein fusions in vivo between the Escherichia coli alkaline phosphatase (APase) and proteins of the facultative photoheterotroph, Rhodobacter sphaeroides. We introduced TnphoA into the genome of R. sphaeroides at a coupled conjugation-transposition frequency of approximately 1 x 10(-6). Fusions giving rise to APase expression, as judged by blue-colony pigmentation when exconjugants were plated on growth medium containing the chromogenic indicator 5-bromo-4-chloro-3-indolyl phosphate, were observed in about 1% of the exconjugants. Numerous, distinguishable mutant phenotypes have been generated by this method, including those which lack the ability to use dimethyl sulfoxide as a terminal electron acceptor during anaerobic respiration, as well as those which are photosynthetically incompetent or altered in pigment synthesis, and others that express resistance to chlorate. The growth and spectral characteristics of several of these mutants, as well as the localization and quantitation of subcellular APase activity under different physiological conditions, have been examined. The presence of TnphoA in the host genome has been confirmed for each mutant analyzed, and specifically tagged DNA fragments containing TnphoA have been identified and localized; cosmids containing R. sphaeroides genomic DNA capable of complementing individual mutants have also been isolated. The usefulness of this approach in studying gene activity in R. sphaeroides is discussed.     

Effect of single mutations on the specificity of Escherichia coli FPG protein for excision of purine lesions from DNA damaged by free radicals

The formamidopyrimidine N-DNA glycosylase (Fpg protein) of Escherichia coli is a DNA repair enzyme that is specific for the removal of purine-derived lesions from DNA damaged by free radicals and other oxidative processes. We investigated the effect of single mutations on the specificity of this enzyme for three purine-derived lesions in DNA damaged by free radicals. These damaging agents generate a multiplicity of base products in DNA, with the yields depending on the damaging agent. Wild type Fpg protein (wt-Fpg) removes 8-hydroxyguanine (8-OH-Gua), 4,6-diamino-5-formamidopyrimidine (FapyAde), and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from damaged DNA with similar specificities. We generated five mutant forms of this enzyme with mutations involving Lys-57-->Gly (FpgK57G), Lys-57-->Arg (FpgK57R), Lys-155-->Ala (FpgK155A), Pro-2-->Gly (FpgP2G), and Pro-2-->Glu (FpgP2E), and purified them to homogeneity. FpgK57G and FpgK57R were functional for removal of FapyAde and FapyGua with a reduced activity when compared with wt-Fpg. The removal of 8-OH-Gua was different in that the specificity of FpgK57G was significantly lower for its removal from irradiated DNA, whereas wt-Fpg, FpgK57G, and FpgK57R excised 8-OH-Gua from H2O2/Fe(III)-EDTA/ascorbic acid-treated DNA with almost the same specificity. FpgK155A and FpgP2G had very low activity and FpgP2E exhibited no activity at all. Michaelis-Menten kinetics of excision was measured and kinetic constants were obtained. The results indicate an important role of Lys-57 residue in the activity of Fpg protein for 8-OH-Gua, but a lesser significant role for formamidopyrimidines. Mutations involving Lys-155 and Pro-2 had a dramatic effect with Pro-2-->Glu leading to complete loss of activity, indicating a significant role of these residues. The results show that point mutations significantly change the specificity of Fpg protein and suggest that point mutations are also expected to change specificities of other DNA repair enzymes.