DNA polymerase beta mRNA and protein expression in Xiphophorus fish

Herein we report Xiphophorus DNA polymerase beta (XiphPolbeta) mRNA and protein expression levels in brain, liver, gill, and testes tissues from Xiphophorus maculatus, Xiphophorus helleri, and Xiphophorus couchianus parental line fish and two different tumor-bearing Xiphophorus interspecies hybrids. Polymerase beta protein levels in the Xiphophorus tissues were measured by Western blot, and mRNA was measured with a quantitative real time RT-PCR method which employed cRNA construction to produce accurate calibration curves. We found significant differences in both mRNA and protein levels between the tumor-bearing hybrid animals and the three parental species. However, there were no significant differences in either mRNA levels or protein expression observed between the parental species. Thus, interspecies hybridization results in dysregulation of Polbeta expression and this may manifest a modulation in DNA repair capability and susceptibility to latent tumorigenesis.     

DNA polymerase X of African swine fever virus: insertion fidelity on gapped DNA substrates and AP lyase activity support a role in base excision repair of viral DNA

DNA polymerase X (pol X) from African swine fever virus (ASFV) is the smallest naturally ocurring DNA-directed DNA polymerase (174 amino acid residues) described so far. Previous biochemical analysis has shown that ASFV pol X is a highly distributive, monomeric enzyme, lacking a proofreading 3'-5' exonuclease. Also, ASFV pol X binds intermediates of the single-nucleotide base excision repair (BER) process, and is able to efficiently repair single-nucleotide gapped DNA. In this work, we perform an extensive kinetic analysis of single correct and incorrect nucleotide insertions by ASFV pol X using different DNA substrates: (i) a primer/template DNA; (ii) a 1nt gapped DNA; (iii) a 5'-phosphorylated 1nt gapped DNA. The results obtained indicate that ASFV pol X exhibits a general preference for insertion of purine deoxynucleotides, especially dGTP opposite template C. Moreover, ASFV pol X shows higher catalytic efficiencies when filling in gapped substrates, which are increased when a phosphate group is present at the 5'-margin of the gap. Interestingly, ASFV pol X misinserts nucleotides with frequencies from 10(-4) to 10(-5), and the insertion fidelity varies depending on the substrate, being more faithful on a phosphorylated 1nt gapped substrate. We have analyzed the capacity of ASFV pol X to act on intermediates of BER repair. Although no lyase activity could be detected on preincised 5'-deoxyribose phosphate termini, ASFV pol X has lyase activity on unincised abasic sites. Altogether, the results support a role for ASFV pol X in reparative BER of damaged viral DNA during ASFV infection.     

An intrinsic 5'-deoxyribose-5-phosphate lyase activity in DNA polymerase beta from Leishmania infantum supports a role in DNA repair

Leishmania infantum is a parasitic protozoan which infects humans. This paper reports the expression in Escherichia coli and purification of the L. infantum gene product (AF182167), as well as its characterization as a DNA polymerase beta (Polbeta)-like, template-dependent DNA repair enzyme, with a metal preference for Mn2+ over Mg2+. As is the case with mammalian Polbeta and DNA polymerase lambda (Pollambda), L. infantum DNA polymerase beta (Li Polbeta) prefers gapped-DNA substrates having a 5'-phosphate end, in agreement with its role in DNA repair reactions. Purified Li Polbeta also displayed a 5'-deoxyribose-5-phosphate (dRP) lyase activity, consistent with a beta-elimination mechanism. The concerted action of dRP lyase and DNA polymerization activities of Li Polbeta on a uracil-containing DNA suggests its participation in "single-nucleotide" base excision repair (BER). Analysis of Li Polbeta DNA polymerization activity at different stages of the L. infantum infective cycle supports a role for Li Polbeta in nuclear DNA repair after the oxidative damage occurring inside the macrophage.     

Deoxyribophosphate lyase activity of mammalian endonuclease VIII-like proteins

Base excision repair (BER) protects cells from nucleobase DNA damage. In eukaryotic BER, DNA glycosylases generate abasic sites, which are then converted to deoxyribo-5'-phosphate (dRP) and excised by a dRP lyase (dRPase) activity of DNA polymerase beta (Polbeta). Here, we demonstrate that NEIL1 and NEIL2, mammalian homologs of bacterial endonuclease VIII, excise dRP by beta-elimination with the efficiency similar to Polbeta. DNA duplexes imitating BER intermediates after insertion of a single nucleotide were better substrates. NEIL1 and NEIL2 supplied dRPase activity in BER reconstituted with dRPase-null Polbeta. Our results suggest a role for NEILs as backup dRPases in mammalian cells.     

Characterization of substrate specificity of plant FatA and FatB acyl-ACP thioesterases

The specificity of plant acyl-acyl carrier protein (ACP) thioesterases is the major determinant of the chain length and level of saturated fatty acids found in most plant tissues. Although these enzymes have been previously characterized from a number of sources, information on kinetic parameters for a wide range of substrates with cloned enzymes is lacking. In the present study the substrate specificity of recombinant FatA thioesterase isoforms from Arabidopsis (AtFatA) and coriander (CsFatA) and FatB from Arabidopsis (AtFatB) have been re-examined with a comprehensive range of substrates including 14:1-ACP and 16:1-ACP. AtFatA displayed the highest catalytic efficiencies (kcat/Km) towards oleoyl-ACP with activities at least 20-fold lower for all other tested substrates and 75-fold lower with palmitoyl-ACP. Both chain length and double bond presence strongly influenced kcat of FatA with minor influence on Km. Arabidopsis FatB substrate specificity was found to differ from previous reports and this difference could be attributed to the influence of ACP structure. FatB activity with palmitoyl-ACP was 2.5-fold higher and the ratio of 16:0-ACP/14:0-ACP hydrolysis was 6.4-fold higher with spinach ACP compared to E. coli ACP. Additionally, the influence of amino acid domains from both AtFatA and AtFatB on their substrate specificity was studied by utilizing a domain-swapping approach. The characterization of the resulting chimeric enzymes pointed to the N-terminus as a determinant of the substrate specificity for both FatA and FatB acyl-ACP thioesterases.     

A survey of the kinetic parameters of class C beta-lactamases. Penicillins.

The interaction between six class C beta-lactamases and various penicillins has been studied. All the enzymes behaved in a very uniform manner. Benzylpenicillin exhibited relatively low kcat. values (14-75 s-1) but low values of Km resulted in high catalytic efficiencies [kcat./Km = 10 X 10(6)-75 X 10(6) M-1.s-1]. The kcat. values for ampicillin were 10-100-fold lower. Carbenicillin, oxacillin cloxacillin and methicillin were very poor substrates, exhibiting kcat. values between 1 x 10(-3) and 0.1 s-1. The Km values were correspondingly small. It could safely be hypothesized that, with all the tested substrates, deacylation was rate-limiting, resulting in acyl-enzyme accumulation.     

Single-turnover kinetic analysis of the mutagenic potential of 8-oxo-7,8-dihydro-2'-deoxyguanosine during gap-filling synthesis catalyzed by human DNA polymerases lambda and beta

In the presence of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) damage, many DNA polymerases exhibit a dual coding potential which facilitates efficient incorporation of matched dCTP or mismatched dATP. This also holds true for the insertion of 8-oxodGTP opposite template bases dC and dA. Employing single-turnover kinetic methods, we examined human DNA polymerase beta and its novel X-family homolog, human DNA polymerase lambda, to determine which nucleotide and template base was preferred when encountering 8-oxodG and 8-oxodGTP, respectively. While DNA polymerase beta preferentially incorporated dCTP over dATP, DNA polymerase lambda did not modulate a preference for either dCTP or dATP when opposite 8-oxodG in single-nucleotide gapped DNA, as incorporation proceeded with essentially equal efficiency and probability. Moreover, DNA polymerase lambda is more efficient than DNA polymerase beta to fill this oxidized single-nucleotide gap. Insertion of 8-oxodGTP by both DNA polymerases lambda and beta occurred predominantly against template dA, thereby reiterating how the asymmetrical design of the polymerase active site differentially accommodated the anti and syn conformations of 8-oxodG and 8-oxodGTP. Although the electronegative oxygen at the C8 position of 8-oxodG may induce DNA structural perturbations, human DNA ligase I was found to effectively ligate the incorporated 8-oxodGMP to a downstream strand, which sealed the nicked DNA. Consequently, the erroneous nucleotide incorporations catalyzed by DNA polymerases lambda and beta as well as the subsequent ligation catalyzed by a DNA ligase during base excision repair are a threat to genomic integrity.     

Alteration of catalytic properties of chymosin by site-directed mutagenesis

Artificial mutations of chymosin by recombinant DNA techniques were generated to analyze the structure--function relationship in this characteristic aspartic proteinase. In order to prepare the mutant enzymes in their active form, we established procedures for purification of correctly refolded prochymosin from inclusion bodies produced in Escherichia coli transformants and for its subsequent activation. Mutagenesis by linker insertion into cDNA produced several mutants with an altered ratio of milk clotting activity to proteolytic activity and a different extent of stability. In addition to these mutants, several mutants with a single amino acid exchange were also constructed by site-directed mutagenesis and kinetic parameters of these mutant enzymes were determined by using synthetic hexa- and octa-peptides as substrates. Exchange of Tyr75 on the flap of the enzyme to Phe caused a marked change of substrate specificity due to the change of kcat or Km, depending on the substrate used. Exchange of Val110 and Phe111 also caused a change of kinetic parameters, which indicates functional involvement of these hydrophobic residues in both the catalytic function and substrate binding. The mutant Lys220----Leu showed a marked shift of the optimum pH to the acidic side for hydrolysis of acid-denatured haemoglobin along with a distinct increase in kcat for the octa-peptide in a wide pH range.     

A novel marker for the platyfish(Xiphophorus maculatus) W chromosome is derived from a Polinton transposon

A consensus sequence,encoding a putative DNA polymerase type B derived from a Polinton transposon,was assembled from the sex determination region of Xiphophorus maculatus.This predicted protein,which is 1,158 aa in length,contains a DNA_pol_B_2 domain and a DTDS motif.The DNA polymerase type B gene has about 10 copies in the haploid X.maculatus genome with one Y-specific copy.Interestingly,it has specific copies on the W chromosome in the X.maculatus Usumacinta strain(sex determination with female heterogam...     

Adduct size limits efficient and error-free bypass across bulky N2-guanine DNA lesions by human DNA polymerase eta

The N2 position of guanine (G) is one of the major sites for DNA modification by various carcinogens. Eight oligonucleotides with varying adduct bulk at guanine N2 were analyzed for catalytic efficiency and fidelity with human DNA polymerase (pol) eta, which is involved in translesion synthesis (TLS). Pol eta effectively bypassed N2-methyl(Me)G, N2-ethyl(Et)G, N2-isobutyl(Ib)G, N2-benzyl(Bz)G, and N2-CH2(2-naphthyl)G but was severely blocked at N2-CH2(9-anthracenyl)G (N2-AnthG) and N2-CH2(6-benzo[a]pyrenyl)G (N2-BPG). Steady-state kinetic analysis showed proportional decreases of kcat/Km in dCTP insertion opposite N2-AnthG and N2-BPG (73 and 320-fold) and also kcat/Km in next-base extension from a C paired with each adduct (15 and 51-fold relative to G). Frequencies of dATP misinsertion and extension beyond mispairs were also proportionally increased (70 and 450-fold; 12 and 44-fold) with N2-AnthG and N2-BPG, indicating the effect of adduct bulk on blocking and misincorporation in TLS by pol eta. N2-AnthG and N2-BPG also greatly decreased the pre-steady-state kinetic burst rate (25 and 125-fold) compared to unmodified G. N2-AnthG decreased dCTP binding affinity (2.6-fold) and increased DNA substrate binding affinity. These results and the small kinetic thio effects (S(p)-dCTPalphaS) suggest that the early steps, possibly conformational change, are interfered with by the bulky adducts. In contrast, human pol delta bypassed adducts effectively up to N2-EtG but was strongly blocked by N2-IbG and larger adducts. We conclude that TLS DNA polymerases may be required for the efficient bypass of pol delta-blocking N2-G adducts bulkier than N2-EtG in human cells, and the bulk size can be a major factor for efficient and error-free bypass at these adducts by TLS DNA polymerases.     

Replacement of an interdomain residue Val39 of Escherichia coli aspartate aminotransferase affects the catalytic competence without altering the substrate specificity of the enzyme

Three mutant Escherichia coli aspartate aminotransferases in which Val39 was changed to Ala, Leu, and Phe by site-directed mutagenesis were prepared and characterized. Among the three mutant and the wild-type enzymes, the Leu39 enzyme had the lowest Km values for dicarboxylic substrates. The Km values of the Ala39 enzyme for dicarboxylates were essentially the same as those of the wild-type (Val39) enzyme. These two mutant enzymes showed essentially the same kcat values for dicarboxylic substrates as did the wild-type enzyme. On the other hand, incorporation of a bulky side-chain at position 39 (Phe39 enzyme) decreased both the affinity (1/Km) and catalytic ability (kcat) toward dicarboxylic substrates. These results show that the position 39 residue is involved in the modulation of both the binding of dicarboxylic substrates to enzyme and the catalytic ability of the enzyme. Although the replacement of Val39 with other residues altered both the kcat and Km values toward various substrates including dicarboxylic and aromatic amino acids and the corresponding oxo acids, it did not alter the ratio of the kcat/Km value of the enzyme toward a dicarboxylic substrate to that for an aromatic substrate. The affinity for aromatic substrates was not affected by changing the residue at position 39. These data indicate that, although the side chain bulkiness of the residue at position 39 correlates well with the activity toward aromatic substrates in the sequence alignment of several aminotransferases [Seville, M., Vincent M.G., & Hahn, K. (1988) Biochemistry 27, 8344-8349], the residue does not seem to be involved in the recognition of aromatic substrates.     

Effect of oxidatively damaged DNA on the active site preorganization during nucleotide incorporation in a high fidelity polymerase from Bacillus stearothermophilus

We study the effect of the oxidative lesion 8-oxoguanine (8oxoG) on the preorganization of the active site for DNA replication in the closed (active) state of the Bacillus fragment (BF), a Klenow analog from Bacillus stearothermophilus. Our molecular dynamics and free energy simulations of explicitly solvated model ternary complexes of BF bound to correct dCTP/incorrect dATP opposite guanine (G) and 8oxoG bases in DNA suggest that the lesion introduces structural and energetic changes at the catalytic site to favor dATP insertion. Despite the formation of a stable Watson-Crick pairing in the 8oxoG:dCTP system, the catalytic geometry is severely distorted to possibly slow down catalysis. Indeed, our calculated free energy landscapes associated with active site preorganization suggest additional barriers to assemble an efficient catalytic site, which need to be overcome during dCTP incorporation opposite 8oxoG relative to that opposite undamaged G. In contrast, the catalytic geometry for the Hoogsteen pairing in the 8oxoG:dATP system is highly organized and poised for efficient nucleotide incorporation via the "two-metal-ion" catalyzed phosphoryl transfer mechanism. However, the free energy calculations suggest that the catalytic geometry during dATP incorporation opposite 8oxoG is considerably less plastic than that during dCTP incorporation opposite G despite a very similar, well organized catalytic site for both systems. A correlation analysis of the dynamics trajectories suggests the presence of significant coupling between motions of the polymerase fingers and the primary distance for nucleophilic attack (i.e., between the terminal primer O3' and the dNTP P(alpha.) atoms) during correct dCTP incorporation opposite undamaged G. This coupling is shown to be disrupted during nucleotide incorporation by the polymerase with oxidatively damaged DNA/dNTP substrates. We also suggest that the lesion affects DNA interactions with key polymerase residues, thereby affecting the enzymes ability to discriminate against non-complementary DNA/dNTP substrates. Taken together, our results provide a unified structural, energetic, and dynamic platform to rationalize experimentally observed relative nucleotide incorporation rates for correct dCTP/incorrect dATP insertion opposite an undamaged/oxidatively damaged template G by BF.     

A novel marker for the platyfish （Xiphophorus maculatus） W chromosome is derived from a Polinton transposon

A consensus sequence,encoding a putative DNA polymerase type B derived from a Polinton transposon,was assembled from the sex determination region of Xiphophorus maculatus.This predicted protein,which is 1,158 as in length,contains a DNA_pol_B_2 domain and a DTDS motif.The DNA polymerase type B gene has about 10 copies in the haploid X.maculatus genome with one Y-specific copy.Interestingly,it has specific copies on the W chromosome in the X.maculatus Usumacinta strain (sex determination with female heterogamety),which represent new markers for this type of sex chromosome in platyfish.This marker with W-and Y-specific copies suggests relationship between different types of gonosomes and allows comparing male and female heterogameties in the platyfish.Further molecular analysis of the DNA polymerase type B gene in X.maculatus will shed new light on the evolution of sex chromosomes in platyfish.     

Altering the conserved nucleotide binding motif in the Salmonella typhimurium MutS mismatch repair protein affects both its ATPase and mismatch binding activities.

The Salmonella typhimurium and Escherichia coli MutS protein is one of several methyl-directed mismatch repair proteins that act together to correct replication errors. MutS is homologous to the Streptococcus pneumoniae HexA mismatch repair protein and to the Duc1 and Rep1 proteins of human and mouse. Homology between the deduced amino acid sequence of both MutS and HexA, and the type A nucleotide binding site consensus sequence, suggested that ATP binding and hydrolysis play a role in their mismatch repair functions. We found that MutS does indeed weakly hydrolyze ATP to ADP and Pi, with a Km of 6 microM and kcat of 0.26. To show that this activity is intrinsic to MutS, we made a site-directed mutation, which resulted in the invariant lysine of the nucleotide binding consensus sequence being changed to an alanine. The mutant MutS allele was unable to complement a mutS::Tn10 mutation in vivo, and was dominant over wild type when present in high copy number. The purified mutant protein had reduced ATPase activity, with the Km affected more severely than the kcat. Like the wild type MutS protein, the mutant protein is able to bind heteroduplex DNA specifically, but the mutant protein does so with a reduced affinity.     

Murine DNA polymerase alpha fills gaps to completion in a direct assay. Altered kinetics of de novo DNA synthesis at single nucleotide gaps

DNA polymerase alpha was studied in a direct gap-filling assay. Using a defined template, DNA synthesis was primed from the M13 17-mer universal primer and blocked by an oligonucleotide hybridized 56 nucleotides downstream of the primer. DNA polymerase alpha filled this gap to completion. A time course of the reaction showed that in 50% of the substrate molecules, gaps were filled to completion within 10 min. In another 35% of the molecules the final nucleotide was lacking after 10 min. This nucleotide was added at a reduced rate, and was not incorporated into all of the molecules even after 6 h. The reduced rate of incorporation of the final nucleotide is reflected in an increased Km for de novo incorporation of one nucleotide at a single nucleotide gap (0.7 microM), as opposed to the Km for de novo incorporation of one nucleotide into singly primed M13 DNA (0.18 microM). DNA polymerase alpha purified from murine cells infected with the parvovirus minute virus of mice, and HeLa cell DNA polymerase alpha 2, exhibited the same kinetics of gap filling as did DNA polymerase alpha purified from uninfected Ehrlich ascites murine tumor cells. T4 DNA polymerase filled gaps to completion in this assay. Escherichia coli DNA polymerase I Klenow fragment quantitatively displaced the downstream oligonucleotide, and extended nascent DNA chains for an additional 100 nucleotides. Nicks and single-nucleotide gaps produced in gap-filling reactions by murine DNA polymerase alpha and T4 DNA polymerase were sealed by T4 DNA ligase.     

Addition of veratryl alcohol oxidase activity to manganese peroxidase by site-directed mutagenesis

Manganese peroxidase and lignin peroxidase are ligninolytic heme-containing enzymes secreted by the white-rot fungus Phanerochaete chrysosporium. Despite structural similarity, these peroxidases oxidize different substrates. Veratryl alcohol is a typical substrate for lignin peroxidase, while manganese peroxidase oxidizes chelated Mn2+. By a single mutation, S168W, we have added veratryl alcohol oxidase activity to recombinant manganese peroxidase expressed in Escherichia coli. The kcat for veratryl alcohol oxidation was 11 s-1, Km for veratryl alcohol approximately 0.49 mM, and Km for hydrogen peroxide approximately 25 microM at pH 2.3. The Km for veratryl alcohol was higher and Km for hydrogen peroxide was lower for this manganese peroxidase mutant compared to two recombinant lignin peroxidase isoenzymes. The mutant retained full manganese peroxidase activity and the kcat was approximately 2.6 x 10(2) s-1 at pH 4.3. Consistent with relative activities with respect to these substrates, Mn2+ strongly inhibited veratryl alcohol oxidation. The single productive mutation in manganese peroxidase suggested that this surface tryptophan residue (W171) in lignin peroxidase is involved in catalysis.     

Partial Purification and Characterization of Soluble Isoform of Butyrylcholinesterase from Rat Intestine

Butyrylcholinesterase (BChE; E.C. 3.1.1.8.) was 260-fold purified from soluble fraction of rat intestine. The enzyme was composed of tetrameric globular form by nonreducing electrophoresis. Optimum pH value was determined as 7.2 after zero buffer extrapolation. Optimum temperature was examined as 37 degrees C after zero time extrapolation. The enzyme showed marked substrate activation with positively charged, acyl-choline substrates. As a measure of catalytic efficiency, kcat/Km values were determined as 16,210, 25,650, and 46,150 for acetylthiocholine (ATCh), propionylthiocholine (PTCh), and butyrylthiocholine (BTCh), respectively. When the catalytic efficiencies are compared, soluble isoform of rat intestinal BChE became increasingly efficient as the size of the acyl portion of the substrate increases; BTCh > PTCh > ATCh. Differently, the enzyme showed substrate inhibition with benzoylcholine (BzCh) and a kcat/Km value of 21,190 was found. Triton X-100 inhibited more efficiently the rat intestinal BChE soluble isoform than it did the human serum BChE.