2-Hydroxy-dATP is incorporated opposite G by Escherichia coli DNA polymerase III resulting in high mutagenicity

Four kinds of oxidatively damaged DNA precursors, 8-hydroxydeoxyguanosine 5′-triphosphate (8-OH-dGTP), 2-hydroxydeoxyadenosine 5′-triphosphate (2-OH-dATP), 5-hydroxydeoxycytidine 5′-triphosphate (5-OH-dCTP) and 5-formyldeoxyuridine 5′-triphosphate (5-CHO-dUTP), were employed in in vitro gap-filling reactions of the supF gene conducted by the Escherichia coli DNA polymerase III holoenzyme, and these treated DNAs were transfected into various E.coli strains. When the manipulated DNAs were transfected into the repair-proficient strain, supF mutants were obtained much more frequently by the purine nucleotides than by the pyrimidine nucleotides (2-OH-dATP > 8-OH-dGTP >> 5-OH-dCTP ~ 5-CHO-dUTP). This result is in contrast to our previous observation that these four oxidatively damaged nucleotides induce chromosomal gene mutations with similar frequencies when incorporated directly into E.coli. 2-OH-dATP elicited G→T transversions, indicating the formation of G•2-OH-dATP pairs. These results demonstrate that 2-OH-dATP was highly mutagenic in this assay system containing the in vitro DNA synthesis by the E.coli replicative DNA polymerase, in addition to in the in vivo assay system reported previously. Slight increases in the mutant frequencies were observed when alkA (for 8-OH-dGTP and 2-OH-dATP) and mutY (for 2-OH-dATP) strains were used as hosts. This is the first report that clearly shows the formation of G•2-OH-dATP pairs.     

In vivo detection and replication studies of α-anomeric lesions of 2′-deoxyribonucleosides

DNA damage, arising from endogenous metabolism or exposure to environmental agents, may perturb the transmission of genetic information by blocking DNA replication and/or inducing mutations, which contribute to the development of cancer and likely other human diseases. Hydroxyl radical attack on the C1′, C3′ and C4′ of 2-deoxyribose can give rise to epimeric 2-deoxyribose lesions, for which the in vivo occurrence and biological consequences remain largely unexplored. Through independent chemical syntheses of all three epimeric lesions of 2′-deoxyguanosine (dG) and liquid chromatography-tandem mass spectrometry analysis, we demonstrated unambiguously the presence of substantial levels of the α-anomer of dG (α-dG) in calf thymus DNA and in DNA isolated from mouse pancreatic tissues. We further assessed quantitatively the impact of all four α-dN lesions on DNA replication in Escherichia coli by employing a shuttle-vector method. We found that, without SOS induction, all α-dN lesions except α-dA strongly blocked DNA replication and, while replication across α-dA was error-free, replicative bypass of α-dC and α-dG yielded mainly C→A and G→A mutations. In addition, SOS induction could lead to markedly elevated bypass efficiencies for the four α-dN lesions, abolished the G→A mutation for α-dG, pronouncedly reduced the C→A mutation for α-dC and triggered T→A mutation for α-dT. The preferential misincorporation of dTMP opposite the α-dNs could be attributed to the unique base-pairing properties of the nucleobases elicited by the inversion of the configuration of the N-glycosidic linkage. Our results also revealed that Pol V played a major role in bypassing α-dC, α-dG and α-dT in vivo. The abundance of α-dG in mammalian tissue and the impact of the α-dNs on DNA replication demonstrate for the first time the biological significance of this family of DNA lesions.     

Synthesis and properties of oligonucleotides containing 5-formyl-2′-deoxycytidine: in vitro DNA polymerase reactions on DNA templates containing 5-formyl-2′-deoxycytidine

Oligodeoxynucleotides (ODNs) containing 5-formyl-2′-deoxycytidine (fC) were synthesized by the phosphoramidite method and subsequent oxidation with sodium periodate. The stabilities of duplexes containing A, G, C or T opposite fC were studied by thermal denaturation. It was found that fC:A, fC:C or fC:T base pairs significantly reduce the thermal stabilities of duplexes. Next, single nucleotide insertion reactions were performed using ODNs containing fC as templates and the Klenow fragment of Escherichia coli DNA polymerase I. It was found that: (i) insertion of dGMP opposite fC appears to be less efficient relative to insertion opposite 5-methyl-2′-deoxycytidine (mC); (ii) dAMP is misincorporated more frequently opposite fC than mC, although the frequency of misincorporation seems to be dependent on the sequence; (iii) TMP is misincorporated more frequently opposite fC than mC. These results suggest that fC may induce the transition mutation C·G→T·A and the transversion mutation C·G→A·T during DNA synthesis.     

A novel mutator of Escherichia coli carrying a defect in the dgt gene, encoding a dGTP triphosphohydrolase

A novel mutator locus in Escherichia coli was identified from a collection of random transposon insertion mutants. Several mutators in this collection were found to have an insertion in the dgt gene, encoding a previously characterized dGTP triphosphohydrolase. The mutator activity of the dgt mutants displays an unusual specificity. Among the six possible base pair substitutions in a lacZ reversion system, the G·C→C·G transversion and A·T→G·C transition are strongly enhanced (10- to 50-fold), while a modest effect (two- to threefold) is also observed for the G·C→A·T transition. Interestingly, a two- to threefold reduction in mutant frequency (antimutator effect) is observed for the G·C→T·A transversion. In the absence of DNA mismatch repair (mutL) some of these effects are reduced or abolished, while other effects remain unchanged. Analysis of these effects, combined with the DNA sequence contexts in which the reversions take place, suggests that alterations of the dGTP pools as well as alterations in the level of some modified dNTP derivatives could affect the fidelity of in vivo DNA replication and, hence, account for the overall mutator effects.     

2-Hydroxy-2'-deoxyadenosine 5'-triphosphate enhances A.T --> C.G mutations caused by 8-hydroxy-2'-deoxyguanosine 5'-triphosphate by suppressing its degradation upon replication in a HeLa extract

The coexistence effects of multiple kinds of oxidized deoxyribonucleotides were examined using an SV40 origin-dependent in vitro replication system with a HeLa extract. Oxidized dGTP and dATP, 8-hydroxy-2'-deoxyguanosine 5'-triphosphate (8-OH-dGTP) and 2-hydroxy-2'-deoxyadenosine 5'-triphosphate (2-OH-dATP), were used in this study. The mutation frequency synergistically increased when the two oxidized deoxyribonucleotides were together in the reaction. 2-OH-dATP enhanced the mutagenicity of 8-OH-dGTP, since the induced mutations were A.T --> C.G transversions. The contribution of the highly error-prone DNA polymerase eta was unlikely, since similar results were observed with an XP-V cell extract. The possible involvement of 2-hydroxyadenine in the complementary (template) strand was excluded on the basis of experiments using plasmids containing 2-hydroxyadenine as templates in the reactions with 8-OH-dGTP. 2-OH-dATP suppressed hydrolysis of 8-OH-dGTP, suggesting that the inhibition of the MTH1 protein played the major role in the enhancement. These results highlight the importance of specific hydrolysis of 8-OH-dGTP for the suppression of its induced mutation.     

Mitochondrial DNA Haplogroups M7b1′2 and M8a Affect Clinical Expression of Leber Hereditary Optic Neuropathy in Chinese Families with the m.11778G→A Mutation

Leber hereditary optic neuropathy (LHON) is the most extensively studied mitochondrial disease, with the majority of the cases being caused by one of three primary mitochondrial DNA (mtDNA) mutations. Incomplete disease penetrance and gender bias are two features of LHON and indicate involvement of additional genetic or environmental factors in the pathogenesis of the disorder. Haplogroups J, K, and H have been shown to influence the clinical expression of LHON in subjects harboring primary mutations in European families. However, whether mtDNA haplogroups would affect the penetrance of LHON in East Asian families has not been evaluated yet. By studying the penetrance of LHON in 1859 individuals from 182 Chinese families (including one from Cambodia) with the m.11778G→A mutation, we found that haplogroup M7b1′2 significantly increases the risk of visual loss, whereas M8a has a protective effect. Analyses of the complete mtDNA sequences from LHON families with m.11778G→A narrow the association of disease expression to m.12811T→C (Y159H) in the NADH dehydrogenase 5 gene (MT-ND5) in haplogroup M7b1′2 and suggest that the specific combination of amino acid changes (A20T-T53I) in the ATP synthase 6 protein (MT-ATP6) caused by m.8584G→A and m.8684C→T might account for the beneficial background effect of M8a. Protein secondary-structure prediction for the MT-ATP6 with the two M8a-specific amino acid changes further supported our inferences. These findings will assist in further understanding the pathogenesis of LHON and guide future genetic counseling in East Asian patients with m.11778G→A.     

Rare missense and synonymous variants in UBE1 are associated with X-linked infantile spinal muscular atrophy

X-linked infantile spinal muscular atrophy (XL-SMA) is an X-linked disorder presenting with the clinical features hypotonia, areflexia, and multiple congenital contractures (arthrogryposis) associated with loss of anterior horn cells and infantile death. To identify the XL-SMA disease gene, we performed large-scale mutation analysis in genes located between markers DXS8080 and DXS7132 (Xp11.3–Xq11.1). This resulted in detection of three rare novel variants in exon 15 of UBE1 that segregate with disease: two missense mutations (c.1617 G→T, p.Met539Ile; c.1639 A→G, p.Ser547Gly) present each in one XL-SMA family, and one synonymous C→T substitution (c.1731 C→T, p.Asn577Asn) identified in another three unrelated families. Absence of the missense mutations was demonstrated for 3550 and absence of the synonymous mutation was shown in 7914 control X chromosomes; therefore, these results yielded statistical significant evidence for the association of the synonymous substitution and the two missense mutations with XL-SMA (p = 2.416 × 10⁻¹⁰, p = 0.001815). We also demonstrated that the synonymous C→T substitution leads to significant reduction of UBE1 expression and alters the methylation pattern of exon 15, implying a plausible role of this DNA element in developmental UBE1 expression in humans. Our observations indicate first that XL-SMA is part of a growing list of neurodegenerative disorders associated with defects in the ubiquitin-proteasome pathway and second that synonymous C→T transitions might have the potential to affect gene expression.     

Direct estimation of the mitochondrial DNA mutation rate in Drosophila melanogaster

Mitochondrial DNA (mtDNA) variants are widely used in evolutionary genetics as markers for population history and to estimate divergence times among taxa. Inferences of species history are generally based on phylogenetic comparisons, which assume that molecular evolution is clock-like. Between-species comparisons have also been used to estimate the mutation rate, using sites that are thought to evolve neutrally. We directly estimated the mtDNA mutation rate by scanning the mitochondrial genome of Drosophila melanogaster lines that had undergone approximately 200 generations of spontaneous mutation accumulation (MA). We detected a total of 28 point mutations and eight insertion-deletion (indel) mutations, yielding an estimate for the single-nucleotide mutation rate of 6.2 × 10⁻⁸ per site per fly generation. Most mutations were heteroplasmic within a line, and their frequency distribution suggests that the effective number of mitochondrial genomes transmitted per female per generation is about 30. We observed repeated occurrences of some indel mutations, suggesting that indel mutational hotspots are common. Among the point mutations, there is a large excess of G→A mutations on the major strand (the sense strand for the majority of mitochondrial genes). These mutations tend to occur at nonsynonymous sites of protein-coding genes, and they are expected to be deleterious, so do not become fixed between species. The overall mtDNA mutation rate per base pair per fly generation in Drosophila is estimated to be about 10× higher than the nuclear mutation rate, but the mitochondrial major strand G→A mutation rate is about 70× higher than the nuclear rate. Silent sites are substantially more strongly biased towards A and T than nonsynonymous sites, consistent with the extreme mutation bias towards A+T. Strand-asymmetric mutation bias, coupled with selection to maintain specific nonsynonymous bases, therefore provides an explanation for the extreme base composition of the mitochondrial genome of Drosophila.     

Two DNA polymerases of Escherichia coli display distinct misinsertion specificities for 2-hydroxy-dATP during DNA synthesis

The insertion specificities of an oxidized dATP analogue, 2-hydroxydeoxyadenosine 5'-triphosphate (2-OH-dATP), were determined using the alpha (catalytic) subunit of Escherichia coli DNA polymerase III and the exonuclease-deficient Klenow fragment of DNA polymerase I. In contrast to our previous observation that mammalian DNA polymerase alpha incorporated the oxidized nucleotide opposite T and C, these two E. coli DNA polymerases incorporated 2-OH-dATP opposite T and G on the DNA template. Steady-state kinetic studies indicated that the alpha subunit incorporated 2-OH-dATP 10 times more frequently opposite T than opposite G. On the other hand, the incorporation of 2-OH-dATP opposite T by the exonuclease-deficient Klenow fragment was 2 orders of magnitude more efficient than that opposite G. These results indicate that the misinsertion specificity of 2-OH-dATP differs between replicative and repair-type DNA polymerases, and provide a biochemical basis for the mutations induced by 2-OH-dATP in E. coli.     

Functional significance of conserved residues in the phosphohydrolase module of Escherichia coli MutT protein

Escherichia coli MutT protein hydrolyzes 8-oxo-7,8-dihydro-2′-dGTP (8-oxo-dGTP) to the monophosphate, thus avoiding the incorporation of 8-oxo-7,8-dihydroguanine (8-oxo-G) into nascent DNA. Bacterial and mammalian homologs of MutT protein share the phosphohydrolase module (MutT: Gly37→Gly59). By saturation mutagenesis of conserved residues in the MutT module, four of the 10 conserved residues (Gly37, Gly38, Glu53 and Glu57) were revealed to be essential to suppress spontaneous A:T→C:G transversion mutation in a mutT^– mutator strain. For the other six residues (Lys39, Glu44, Thr45, Arg52, Glu56 and Gly59), many positive mutants which can suppress the spontaneous mutation were obtained; however, all of the positive mutants for Glu44 and Arg52 either partially or inefficiently suppressed the mutation, indicating that these two residues are also important for MutT function. Several positive mutants for Lys39, Thr45, Glu56 and Gly59 efficiently decreased the elevated spontaneous mutation rate, as seen with the wild-type, hence, these four residues are non-essential for MutT function. As Lys38 and Glu55 in human MTH1, corresponding to the non-essential residues Lys39 and Glu56 in MutT, could not be replaced by any other residue without loss of function, different structural features between the two modules of MTH1 and MutT proteins are evident.     

Replicative DNA Polymerase δ but Not ε Proofreads Errors in Cis and in Trans

It is now well established that in yeast, and likely most eukaryotic organisms, initial DNA replication of the leading strand is by DNA polymerase ε and of the lagging strand by DNA polymerase δ. However, the role of Pol δ in replication of the leading strand is uncertain. In this work, we use a reporter system in Saccharomyces cerevisiae to measure mutation rates at specific base pairs in order to determine the effect of heterozygous or homozygous proofreading-defective mutants of either Pol ε or Pol δ in diploid strains. We find that wild-type Pol ε molecules cannot proofread errors created by proofreading-defective Pol ε molecules, whereas Pol δ can not only proofread errors created by proofreading-defective Pol δ molecules, but can also proofread errors created by Pol ε-defective molecules. These results suggest that any interruption in DNA synthesis on the leading strand is likely to result in completion by Pol δ and also explain the higher mutation rates observed in Pol δ-proofreading mutants compared to Pol ε-proofreading defective mutants. For strains reverting via AT→GC, TA→GC, CG→AT, and GC→AT mutations, we find in addition a strong effect of gene orientation on mutation rate in proofreading-defective strains and demonstrate that much of this orientation dependence is due to differential efficiencies of mispair elongation. We also find that a 3′-terminal 8 oxoG, unlike a 3′-terminal G, is efficiently extended opposite an A and is not subject to proofreading. Proofreading mutations have been shown to result in tumor formation in both mice and humans; the results presented here can help explain the properties exhibited by those proofreading mutants.     

Crystal structure of parallel G-quadruplex formed by the two-repeat ALS- and FTD-related GGGGCC sequence

The hexanucleotide repeat expansion, GGGGCC (G4C2), within the first intron of the C9orf72 gene is known to be the most common genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The G4C2 repeat expansions, either DNA or RNA, are able to form G-quadruplexes which induce toxicity leading to ALS/FTD. Herein, we report a novel crystal structure of d(G4C2)₂ that self-associates to form an eight-layer parallel tetrameric G-quadruplex. Two d(G4C2)₂ associate together as a parallel dimeric G-quadruplex which folds into a tetramer via 5′-to-5′ arrangements. Each dimer consists of four G-tetrads connected by two CC propeller loops. Especially, the 3′-end cytosines protrude out and form C·C+•C·C+/ C·C•C·C+ quadruple base pair or C•C·C+ triple base pair stacking on the dimeric block. Our work sheds light on the G-quadruplexes adopted by d(G4C2) and yields the invaluable structural details for the development of small molecules to tackle neurodegenerative diseases, ALS and FTD.     

Nearest-neighbor thermodynamics of deoxyinosine pairs in DNA duplexes

Nearest-neighbor thermodynamic parameters of the ‘universal pairing base’ deoxyinosine were determined for the pairs I·C, I·A, I·T, I·G and I·I adjacent to G·C and A·T pairs. Ultraviolet absorbance melting curves were measured and non-linear regression performed on 84 oligonucleotide duplexes with 9 or 12 bp lengths. These data were combined with data for 13 inosine containing duplexes from the literature. Multiple linear regression was used to solve for the 32 nearest-neighbor unknowns. The parameters predict the T_m for all sequences within 1.2°C on average. The general trend in decreasing stability is I·C > I·A > I·T ≈ I· G > I·I. The stability trend for the base pair 5′ of the I·X pair is G·C > C·G > A·T > T·A. The stability trend for the base pair 3′ of I·X is the same. These trends indicate a complex interplay between H-bonding, nearest-neighbor stacking, and mismatch geometry. A survey of 14 tandem inosine pairs and 8 tandem self-complementary inosine pairs is also provided. These results may be used in the design of degenerate PCR primers and for degenerate microarray probes.     

RAD51 135G-->C modifies breast cancer risk among BRCA2 mutation carriers: results from a combined analysis of 19 studies

RAD51 is an important component of double-stranded DNA–repair mechanisms that interacts with both BRCA1 and BRCA2. A single-nucleotide polymorphism (SNP) in the 5′ untranslated region (UTR) of RAD51, 135G→C, has been suggested as a possible modifier of breast cancer risk in BRCA1 and BRCA2 mutation carriers. We pooled genotype data for 8,512 female mutation carriers from 19 studies for the RAD51 135G→C SNP. We found evidence of an increased breast cancer risk in CC homozygotes (hazard ratio [HR] 1.92 [95% confidence interval {CI} 1.25–2.94) but not in heterozygotes (HR 0.95 [95% CI 0.83–1.07]; P=.002, by heterogeneity test with 2 degrees of freedom [df]). When BRCA1 and BRCA2 mutation carriers were analyzed separately, the increased risk was statistically significant only among BRCA2 mutation carriers, in whom we observed HRs of 1.17 (95% CI 0.91–1.51) among heterozygotes and 3.18 (95% CI 1.39–7.27) among rare homozygotes (P=.0007, by heterogeneity test with 2 df). In addition, we determined that the 135G→C variant affects RAD51 splicing within the 5′ UTR. Thus, 135G→C may modify the risk of breast cancer in BRCA2 mutation carriers by altering the expression of RAD51. RAD51 is the first gene to be reliably identified as a modifier of risk among BRCA1/2 mutation carriers.     

Excision of 8-oxoguanine within clustered damage by the yeast OGG1 protein

Clustered damages are formed in DNA by ionising radiation and radiomimetic anticancer agents and are thought to be biologically severe. 7,8-dihydro-8-oxoguanine (8-oxoG), a major DNA damage resulting from oxidative attack, is highly mutagenic leading to a high level of G·C→T·A transversions if not previously excised by OGG1 DNA glycosylase/AP lyase proteins in eukaryotes. However, 8-oxoG within clustered DNA damage may present a challenge to the repair machinery of the cell. The ability of yeast OGG1 to excise 8-oxoG was determined when another type of damage [dihydrothymine, uracil, 8-oxoG, abasic (AP) site or various types of single-strand breaks (SSBs)] is present on the complementary strand 1, 3 or 5 bases 5′ or 3′ opposite to 8-oxoG. Base damages have little or no influence on the excision of 8-oxoG by yeast OGG1 (yOGG1) whereas an AP site has a strong inhibitory effect. Various types of SSBs, obtained using either oligonucleotides with 3′- and 5′-phosphate termini around a gap or through conversion of an AP site with either endonuclease III or human AP endonuclease 1, strongly inhibit excision of 8-oxoG by yOGG1. Therefore, this large inhibitory effect of an AP site or a SSB may minimise the probability of formation of a double-strand break in the processing of 8-oxoG within clustered damages.     

Human Coding Synonymous Single Nucleotide Polymorphisms at Ramp Regions of mRNA Translation

According to the ramp model of mRNA translation, the first 50 codons favor rare codons and have slower speed of translation. This study aims to detect translational selection on coding synonymous single nucleotide polymorphisms (sSNP) to support the ramp theory. We investigated fourfold degenerate site (FFDS) sSNPs with A↔G or C↔T substitutions in human genome for distribution bias of synonymous codons (SC), grouped by CpG or non-CpG sites. Distribution bias of sSNPs between the 3^rd ∼50^th codons and the 51^st ∼ remainder codons at non-CpG sites were observed. In the 3^rd ∼50^th codons, G→A sSNPs at non-CpG sites are favored than A→G sSNPs [P = 2.89×10⁻³], and C→T at non-CpG sites are favored than T→C sSNPs [P = 8.50×10⁻³]. The favored direction of SC usage change is from more frequent SCs to less frequent SCs. The distribution bias is more obvious in synonymous substitutions CG(G→A), AC(C→T), and CT(C→T). The distribution bias of sSNPs in human genome, i.e. frequent SCs to less frequent SCs is favored in the 3^rd ∼50^th codons, indicates translational selection on sSNPs in the ramp regions of mRNA templates.     

A novel Nudix hydrolase for oxidized purine nucleoside triphosphates encoded by ORFYLR151c (PCD1 gene) in Saccharomyces cerevisiae

A search for candidates for a functional homologue of Escherichia coli MutT in yeast Saccharomyces cerevisiae was made in the NCBI-BLAST database using the Nudix box, a short amino acid sequence conserved among E.coli MutT, Pseudomonoas vulgaris MutT, and human, rat and mouse MTH1. Among five candidates, we focused on the open reading frame YLR151c, because it had a region with ~76% similarity to the N-terminal half of MutT including the Nudix box. We thus evaluated the ability of YLR151c as a functional homologue of E.coli MutT in S.cerevisiae. Expression of YLR151c was able to suppress the transversion from A:T to C:G caused by misincorporation of the oxidized nucleotide 8-oxo-dGTP in the E.coli mutT-deficient strain. The disruption of the YLR151c in yeast strain caused ~14-fold increase in the frequency of spontaneous mutation compared to the wild type. Additionally, biochemical analysis indicated that GST-YLR151c fusion protein possessed pyrophosphatase activity for both 7,8-dihydro-8-oxo-2′-deoxyguanosine triphosphate (8-oxo-dGTP) and 1,2-dihydro-2-hydroxy-2′-deoxyadenosine triphosphate (2-OH-dATP). The specific activity of GST-YLR151c for 8-oxo-dGTP was 5.6 × 10⁻³ μM⁻¹ s⁻¹, which was similar to that of RibA, a backup enzyme for MutT in E.coli, but was 150-fold lower than that of hMTH1. From these results, we conclude that YLR151c has an ability to prevent spontaneous mutagenesis via sanitization of oxidized nucleotides, and that it may be the functional homologue of E.coli MutT in S.cerevisiae.     

MTH1, an oxidized purine nucleoside triphosphatase, prevents the cytotoxicity and neurotoxicity of oxidized purine nucleotides

In human and rodent cells, MTH1, an oxidized purine nucleoside triphosphatase, efficiently hydrolyzes oxidized dGTP, GTP, dATP and ATP such as 2'-deoxy-8-oxoguanosine triphosphate (8-oxo-dGTP) and 2'-deoxy-2-hydroxyadenosine triphosphate (2-OH-dATP) in nucleotide pools, thus avoiding their incorporation into DNA or RNA. MTH1 is expressed in postmitotic neurons as well as in proliferative tissues, and it is localized both in the mitochondria and nucleus, thus suggesting that MTH1 plays an important role in the prevention of the mutagenicity and cytotoxicity of such oxidized purines as 8-oxoG which are known to accumulate in the cellular genome. Our recent studies with MTH1-deficient mice or cells revealed that MTH1 efficiently minimizes accumulation of 8-oxoG in both nuclear and mitochondrial DNA in the mouse brain as well as in cultured cells, thus contributing to the protection of the brain from oxidative stress.     

Thermoadaptation-Directed Enzyme Evolution in an Error-Prone Thermophile Derived from Geobacillus kaustophilus HTA426

Thermostability is an important property of enzymes utilized for practical applications because it allows long-term storage and use as catalysts. In this study, we constructed an error-prone strain of the thermophile Geobacillus kaustophilus HTA426 and investigated thermoadaptation-directed enzyme evolution using the strain. A mutation frequency assay using the antibiotics rifampin and streptomycin revealed that G. kaustophilus had substantially higher mutability than Escherichia coli and Bacillus subtilis. The predominant mutations in G. kaustophilus were A · T→G · C and C · G→T · A transitions, implying that the high mutability of G. kaustophilus was attributable in part to high-temperature-associated DNA damage during growth. Among the genes that may be involved in DNA repair in G. kaustophilus, deletions of the mutSL, mutY, ung, and mfd genes markedly enhanced mutability. These genes were subsequently deleted to construct an error-prone thermophile that showed much higher (700- to 9,000-fold) mutability than the parent strain. The error-prone strain was auxotrophic for uracil owing to the fact that the strain was deficient in the intrinsic pyrF gene. Although the strain harboring Bacillus subtilis pyrF was also essentially auxotrophic, cells became prototrophic after 2 days of culture under uracil starvation, generating B. subtilis PyrF variants with an enhanced half-denaturation temperature of >10°C. These data suggest that this error-prone strain is a promising host for thermoadaptation-directed evolution to generate thermostable variants from thermolabile enzymes.     

Human replication protein A (RPA) binds a primer-template junction in the absence of its major ssDNA-binding domains

The human nuclear single-stranded (ss) DNA- binding protein, replication protein A (RPA), is a heterotrimer consisting of three subunits: p70, p32 and p14. The protein–DNA interaction is mediated by several DNA-binding domains (DBDs): two major (A and B, also known as p70A and p70B) and several minor (C and D, also known as p70C and p32D, and, presumably, by p70N). Here, using crosslinking experiments, we investigated an interaction of RPA deletion mutants containing a subset of the DBDs with partial DNA duplexes containing 5′-protruding ssDNA tails of 10, 20 and 30 nt. The crosslinks were generated using either a ‘zero-length’ photoreactive group (4-thio-2′-deoxyuridine-5′-monophosphate) embedded in the 3′ end of the DNA primer, or a group connected to the 3′ end by a lengthy linker (5-{N-[N-(4-azido-2,5-difluoro-3- chloropyridine-6-yl)-3-aminopropionyl]-trans-3-aminopropenyl-1}-2′-deoxyuridine-5′-monophosphate). In the absence of two major DBDs, p70A and p70B, the RPA trimerization core (p70C·p32D·p14) was capable of correctly recognizing the primer– template junction and adopting an orientation similar to that in native RPA. Both p70C and p32D contributed to this recognition. However, the domain contribution differed depending on the size of the ssDNA. In contrast with the trimerization core, the RPA dimerization core (p32D·p14) was incapable of detectably recognizing the DNA- junction structures, suggesting an orchestrating role for p70C in this process.