Deletion loop mutagenesis: a novel method for the construction of point mutations using deletion mutants

Deletion loop mutagenesis is a new, general method for site-directed mutagenesis that allows point mutations to the introduced within a sequence of DNA defined by a previously isolated deletion mutant. Wild type and deletion mutant DNA are cloned into a bacterial plasmid and each is cleaved with a different single cut restriction enzyme. Heteroduplexes are formed between the two DNAs to produce circular molecules containing a nick in each strand and a single-stranded deletion loop. The deletion loops are mutagenised using sodium bisulphite and the DNA transfected directly into a uracil repair deficient strain of Escherichia coli. Up to half of the resultant clones contain DNA produced by replication of the wild-type length strand and bear mutations exclusively within the target area. An example is given in which a deletion mutant lacking 21 nucleotides from the region coding for SV40 large-T was used. Eight of the possible nine target cytosine residues were mutagenised. The method described is specific, efficient and simple.     

UNG-dependent correction of molecular heteroduplexes of M13 phage DNA in Escherichia coli cells

Correction of heteroduplex DNA obtained by hybridization of uracil-containing single-stranded M13mp18 phage DNA and "mutant" synthetic oligonucleotide with deletion of cytosine in SalGI site was studied in ung+ and ung- E. coli strains. Uracil-containing DNA was prepared after growth of phage in an E. coli strain dut- ung-. The DNA was hybridized with "mutant" oligonucleotide then complementary DNA chain was synthesized by T4 DNA polymerase. Ung+ and ung- E. coli cells were transformed by DNA. In all experiments mutation frequency in ung+ was higher than in ung- cells (approximately 6-fold) and reached 11-50%. Absolute number of mutants was higher in ung+ cells. The results indicate that high level of mutagenesis depends on uracil repair system polarizing the correction of heteroduplex DNA.     

Consensus sequences for good and poor removal of uracil from double stranded DNA by uracil-DNA glycosylase.

We have purified uracil DNA-glycosylase (UDG) from calf thymus 32,000-fold and studied its biochemical properties, including sequence specificity. The enzyme is apparently closely related to human UDG, since it was recognised by a polyclonal antibody directed towards human UDG. SDS-PAGE and western analysis indicate an apparent M(r) = 27,500. Bovine UDG has a 1.7-fold preference for single stranded over double stranded DNA as a substrate. Sequence specificity for uracil removal from dsDNA was examined for bovine and Escherichia coli UDG, using DNA containing less than one dUMP residue per 100 nucleotides and synthetic oligonucleotides containing one dUMP residue. Comparative studies involving about 40 uracil sites indicated similar specificities for both UDGs. We found more than a 10-fold difference in rates of uracil removal between different sequences. 5'-G/CUT-3' and 5'-G/CUG/C-3' were consensus sequences for poor repair whereas 5'-A/TUAA/T-3' was a consensus for good repair. Sequence specificity was verified in double stranded oligonucleotides, but not in single stranded ones, suggesting that the structure of the double stranded DNA helix has influence on sequence specificity. Rate of uracil removal appeared to be slightly faster from U:A base pairs as compared to U:G mis-matches. The results indicate that sequence specific repair may be a determinant to be considered in mutagenesis.     

Induction of SOS functions in Escherichia coli by lesions resulting from incorporation of 5-bromouracil into DNA

Lesions induced by 5-bromouracil (BU), after its incorporation into DNA, led to effective induction of prophage lambda and W reactivation (or BU reactivation). Prophage induction due to incorporated BU occurred only with the wild-type prophage, and not for the lambda c1857 mutant with a thermosensitive repressor. Antipain, a protease inhibitor, inhibited wild-type prophage induction 70-90%. This indicates that BU-induced lesions may induce the SOS repair system. The finding that such lesions provoke BU reactivation permits the inference that BU-induced mutagenesis also proceeds via involvement of the error-prone repair system, and not directly as a result of base-pairing errors. Genetic evidence suggests that induction of the SOS repair system as a result of incorporation of BU into DNA is linked to the subsequent appearance of uracil residues and apyrimidinic sites, resulting from dehalogenation of incorporated BU. Apyrimidinic sites appear to be more effective than uracil residues in induction of the SOS system.     

Dehydration, deamination and enzymatic repair of cytosine glycols from oxidized poly(dG-dC) and poly(dI-dC)

Cytosine glycols (5,6-dihydroxy-5,6-dihydrocytosine) are initial products of cytosine oxidation. Because these products are not stable, virtually all biological studies have focused on the stable oxidation products of cytosine, including 5-hydroxycytosine, uracil glycols and 5-hydroxyuracil. Previously, we reported that the lifetime of cytosine glycols was greatly enhanced in double-stranded DNA, thus implicating these products in DNA repair and mutagenesis. In the present work, cytosine and uracil glycols were generated in double-stranded alternating co-polymers by oxidation with KMnO₄. The half-life of cytosine glycols in poly(dG-dC) was 6.5 h giving a ratio of dehydration to deamination of 5:1. At high substrate concentrations, the excision of cytosine glycols from poly(dG-dC) by purified endonuclease III was comparable to that of uracil glycols, whereas the excision of these substrates was 5-fold greater than that of 5-hydroxycytosine. Kinetic studies revealed that the V_max was several fold higher for the excision of cytosine glycols compared to 5-hydroxycytosine. In contrast to cytosine glycols, uracil glycols did not undergo detectable dehydration to 5-hydroxyuracil. Replacing poly(dG-dC) for poly(dI-dC) gave similar results with respect to the lifetime and excision of cytosine glycols. This work demonstrates the formation of cytosine glycols in DNA and their removal by base excision repair.     

灵芝尿嘧啶营养缺陷型菌株的筛选和分子鉴定

【背景】营养缺陷型是一种应用广泛的分子标记,但是目前在灵芝中还未有研究和应用报道。【目的】为灵芝遗传转化研究、杂交育种和菌种鉴别提供亲本材料和技术支持。【方法】采用紫外光诱变、单单杂交、孢子单核化的方法从灵芝单核体菌株出发得到尿嘧啶营养缺陷型双核体菌株。【结果】获得8株稳定的尿嘧啶营养缺陷型单核体突变菌株和7株尿嘧啶营养缺陷型双核体菌株。【结论】灵芝尿嘧啶营养缺陷型菌株在添加外源营养物的基础上可恢复正常生长,可以为灵芝遗传转化体系的构建和灵芝育种提供材料。     

Identification of uracil as a major lesion in E. coli DNA following the incorporation of 5-bromouracil, and some of the accompanying effects

Cultivation of E. coli cells in the presence of 5-bromodeoxyuridine (BUdR) leads to formation of lesions in the cellular DNA which affect its secondary structure, as reflected by changes in temperature profiles. Such DNA contains single-stranded regions susceptible to endonuclease S1. One of the major sources of the BU-induced lesions appears to be dehalogenation of incorporated 5-bromouracil (BU) residues, with accompanying formation of uracil. The presence of uracil residues in such DNA was demonstrated directly by chromatography of hydrolyzates, and by the susceptibility of such residues to uracil-DNA glycosylase. The number of uracil residues was dependent on the extent of damage in the DNA, and decreased during the DNA repair that accompanied reactivation of bromouracil-inactivated cells. Dehalogenation of incorporated BU presumably results in formation of apyrimidinic sites by uracil-DNA glycosylase, and then single-strand nicks either by AP-endonuclease and/or dehalogenation. The findings are relevant to the mechanism of BU-induced mutagenesis.     

Vaccinia virus uracil DNA glycosylase has an essential role in DNA synthesis that is independent of its glycosylase activity: catalytic site mutations reduce virulence but not virus replication in cultured cells

Previous findings that the vaccinia virus uracil DNA glycosylase is required for virus DNA replication, coupled with an inability to isolate a mutant with an active site substitution in the glycosylase gene, were surprising, as such enzymes function in DNA repair and bacterial, yeast, and mammalian null mutants are viable. To further study the role of the viral protein, we constructed recombinant vaccinia viruses with single or double mutations (D68N and H181L) in the uracil DNA glycosylase conserved catalytic site by using a complementing cell line that constitutively expresses the viral enzyme. Although these mutations abolished uracil DNA glycosylase activity, they did not prevent viral DNA replication or propagation on a variety of noncomplementing cell lines or human primary skin fibroblasts. In contrast, replication of a uracil DNA glycosylase deletion mutant occurred only in the complementing cell line. Therefore, the uracil DNA glycosylase has an essential role in DNA replication that is independent of its glycosylase activity. Nevertheless, the conservation of the catalytic site in all poxvirus orthologs suggested an important role in vivo. This idea was confirmed by the decreased virulence of catalytic-site mutants when administered by the intranasal route to mice.     

C --> T mutagenesis and gamma-radiation sensitivity due to deficiency in the Smug1 and Ung DNA glycosylases

The most common genetic change in aerobic organisms is a C:G to T:A mutation. C --> T transitions can arise through spontaneous hydrolytic deamination of cytosine to give a miscoding uracil residue. This is also a frequent DNA lesion induced by oxidative damage, through exposure to agents such as ionizing radiation, or from endogenous sources that are implicated in the aetiology of degenerative diseases, ageing and cancer. The Ung and Smug1 enzymes excise uracil from DNA to effect repair in mammalian cells, and gene-targeted Ung(-/-) mice exhibit a moderate increase in genome-wide spontaneous mutagenesis. Here, we report that stable siRNA-mediated silencing of Smug1 in mouse embryo fibroblasts also generates a mutator phenotype. However, an additive 10-fold increase in spontaneous C:G to T:A transitions in cells deficient in both Smug1 and Ung demonstrates that these enzymes have distinct and nonredundant roles in suppressing C --> T mutability at non-CpG sites. Such cells are also hypersensitive to ionizing radiation, and reveal a role of Smug1 in the repair of lesions generated by oxidation of cytosine.     

Repair of the mutagenic DNA oxidation product, 5-formyluracil

The oxidation of the thymine methyl group can generate 5-formyluracil (FoU). Template FoU residues are known to miscode, generating base substitution mutations. The repair of the FoU lesion is therefore important in minimizing mutations induced by DNA oxidation. We have studied the repair of FoU in synthetic oligonucleotides when paired with A and G. In E. coli cell extract, the repair of FoU is four orders of magnitude lower than the repair of U and is similar for both FoU:A and FoU:G base pairs. In HeLa nuclear extract, the repair of FoU:A is similarly four orders of magnitude lower than the repair of uracil, although the FoU:G lesion is repaired 10 times more efficiently than FoU:A. The FoU:G lesion is shown to be repaired by E. coli mismatch uracil DNA glycosylase (Mug), thermophile mismatch thymine DNA glycosylase (Tdg), mouse mismatch thymine DNA glycosylase (mTDG) and human methyl-CpG-binding thymine DNA glycosylase (MBD4), whereas the FoU:A lesion is repaired only by Mug and mTDG. The repair of FoU relative to the other pyrimidines examined here in human cell extract differs from the substrate preferences of the known glycosylases, suggesting that additional, and as yet unidentified glycosylases exist in human cells to repair the FoU lesion. Indeed, as observed in HeLa nuclear extract, the repair of mispaired FoU derived from misincorporation of dGMP across from template FoU could promote rather than minimize mutagenesis. The pathways by which this important lesion is repaired in human cells are as yet unexplained, and are likely to be complex.     

Roles of endonuclease V, uracil-DNA glycosylase, and mismatch repair in Bacillus subtilis DNA base-deamination-induced mutagenesis

The disruption of ung, the unique uracil-DNA-glycosylase-encoding gene in Bacillus subtilis, slightly increased the spontaneous mutation frequency to rifampin resistance (Rif(r)), suggesting that additional repair pathways counteract the mutagenic effects of uracil in this microorganism. An alternative excision repair pathway is involved in this process, as the loss of YwqL, a putative endonuclease V homolog, significantly increased the mutation frequency of the ung null mutant, suggesting that Ung and YwqL both reduce the mutagenic effects of base deamination. Consistent with this notion, sodium bisulfite (SB) increased the Rif(r) mutation frequency of the single ung and double ung ywqL strains, and the absence of Ung and/or YwqL decreased the ability of B. subtilis to eliminate uracil from DNA. Interestingly, the Rif(r) mutation frequency of single ung and mutSL (mismatch repair [MMR] system) mutants was dramatically increased in a ung knockout strain that was also deficient in MutSL, suggesting that the MMR pathway also counteracts the mutagenic effects of uracil. Since the mutation frequency of the ung mutSL strain was significantly increased by SB, in addition to Ung, the mutagenic effects promoted by base deamination in growing B. subtilis cells are prevented not only by YwqL but also by MMR. Importantly, in nondividing cells of B. subtilis, the accumulations of mutations in three chromosomal alleles were significantly diminished following the disruption of ung and ywqL. Thus, under conditions of nutritional stress, the processing of deaminated bases in B. subtilis may normally occur in an error-prone manner to promote adaptive mutagenesis.     

The spectrum of spontaneous mutations in a Saccharomyces cerevisiae uracil-DNA-glycosylase mutant limits the function of this enzyme to cytosine deamination repair.

Uracil-DNA-glycosylase has been proposed to function as the first enzyme in strand-directed mismatch repair in eukaryotic organisms, through removal of uracil from dUMP residues periodically inserted into the DNA during DNA replication (Aprelikova, O. N., V. M. Golubovskaya, T. A. Kusmin, and N. V. Tomilin, Mutat. Res. 213:135-140, 1989). This hypothesis was investigated with Saccharomyces cerevisiae. Mutation frequencies and spectra were determined for an ung1 deletion strain in the target SUP4-o tRNA gene by using a forward selection scheme. Mutation frequencies in the SUP4-o gene increased about 20-fold relative to an isogenic wild-type S. cerevisiae strain, and the mutator effect was completely suppressed in the ung1 deletion strain carrying the wild-type UNG1 gene on a multicopy plasmid. Sixty-nine independently derived mutations in the SUP4-o gene were sequenced. All but five of these were due to GC----AT transitions. From this analysis, we conclude that the mutator phenotype of the ung1 deletion strain is the result of a failure to repair spontaneous cytosine deamination events occurring frequently in S. cerevisiae and that the UNG1 gene is not required for strand-specific mismatch repair in S. cerevisiae.     

CRISPR/Cas9-mediated mutagenesis at microhomologous regions of human mitochondrial genome

Genetic manipulation of mitochondrial DNA(mtDNA) could be harnessed for deciphering the gene function of mitochondria; it also acts as a promising approach for the therapeutic correction of pathogenic mutation in mtDNA. However, there is still a lack of direct evidence showing the edited mutagenesis within human mtDNA by clustered regularly interspaced short palindromic repeats-associated protein 9(CRISPR/Cas9). Here, using engineered CRISPR/Cas9, we observed numerous insertion/deletion(InDel) events at several mtDNA microhomologous regions, which were triggered specifically by double-strand break(DSB)lesions within mtDNA. InDel mutagenesis was significantly improved by sgRNA multiplexing and a DSB repair inhibitor,iniparib, demonstrating the evidence of rewiring DSB repair status to manipulate mtDNA using CRISPR/Cas9. These findings would provide novel insights into mtDNA mutagenesis and mitochondrial gene therapy for diseases involving pathogenic mtDNA.     

Increased uracil insertion in DNA is cytotoxic and increases the frequency of mutation,double strand break formation and VSG switching in Trypanosoma brucei

Deoxyuridine 5′-triphosphate pyrophosphatase (dUTPase) and uracil-DNA glycosylase (UNG) are key enzymes involved in the control of the presence of uracil in DNA. While dUTPase prevents uracil misincorporation by removing dUTP from the deoxynucleotide pool, UNG excises uracil from DNA as a first step of the base excision repair pathway (BER). Here, we report that strong down-regulation of dUTPase in UNG-deficient Trypanosoma brucei cells greatly impairs cell viability in both bloodstream and procyclic forms, underscoring the extreme sensitivity of trypanosomes to uracil in DNA. Depletion of dUTPase activity in the absence of UNG provoked cell cycle alterations, massive dUTP misincorporation into DNA and chromosomal fragmentation. Overall, trypanosomatid cells that lack dUTPase and UNG activities exhibited greater proliferation defects and DNA damage than cells deficient in only one of these activities. To determine the mutagenic consequences of uracil in DNA, mutation rates and spectra were analyzed in dUTPase-depleted cells in the presence of UNG activity. These cells displayed a spontaneous mutation rate 9-fold higher than the parental cell line. Base substitutions at A:T base pairs and deletion frequencies were both significantly enhanced which is consistent with the generation of mutagenic AP sites and DNA strand breaks. The increase in strand breaks conveyed a concomitant increase in VSG switching in vitro. The low tolerance of T. brucei to uracil in DNA emphasizes the importance of uracil removal and regulation of intracellular dUTP pool levels in cell viability and genetic stability and suggests potential strategies to compromise parasite survival.     

Oligonucleotide-directed gene repair in wheat using a transient plasmid gene repair assay system

Oligonucleotide-directed gene repair is a potential technique for agricultural trait modification in economically important crops. However, large variation in the repair frequencies among the scientific reports indicates that there are many factors influencing the repair process. We report here a transient assay system using GFP as a reporter for testing the efficiency of plasmid DNA repair in cultured wheat cells. This assay showed that osmotic medium supplemented with 2,4-D increased the oligo-targeting frequency, and that the repair of a point mutation was more efficient than repair of a single base deletion mutation in cultured scutellum cells of immature wheat embryos. This study provides the first evidence that oligonucleotide-directed mutagenesis is applicable to regenerable cultured wheat scutellum cells.     

REV3 is required for spontaneous but not methylation damage-induced mutagenesis of Saccharomyces cerevisiae cells lacking O6-methylguanine DNA methyltransferase

O6-methylguanine (O6-MeG) DNA methyltransferase (MTase) removes the methyl group from a DNA lesion and directly restores DNA structure. It has been shown previously that bacterial and yeast cells lacking such MTase activity are not only sensitive to killing and mutagenesis by DNA methylating agents, but also exhibit an increased spontaneous mutation rate. In order to understand molecular mechanisms of endogenous DNA alkylation damage and its effects on mutagenesis, we determined the spontaneous mutational spectra of the SUP4-o gene in various Saccharomyces cerevisiae strains. To our surprise, the mgt1 mutant deficient in DNA repair MTase activity exhibited a significant increase in G:C-->C:G transversions instead of the expected G:C-->A:T transition. Its mutational distribution strongly resembles that of the rad52 mutant defective in DNA recombinational repair. The rad52 mutational spectrum has been shown to be dependent on a mutagenesis pathway mediated by REV3. We demonstrate here that the mgt1 mutational spectrum is also REV3-dependent and that the rev3 deletion offsets the increase of the spontaneous mutation rate seen in the mgt1 strains. These results indicate that the eukaryotic mutagenesis pathway is directly involved in cellular processing of endogenous DNA alkylation damage possibly by the translesion bypass of lesions at the cost of G:C-->C:G transversion mutations. However, the rev3 deletion does not affect methylation damage-induced killing and mutagenesis of the mgt1 mutant, suggesting that endogenous alkyl lesions may be different from O6-MeG.     

Human cytomegalovirus uracil DNA glycosylase is required for the normal temporal regulation of both DNA synthesis and viral replication.

Human cytomegalovirus (CMV) encodes a gene, UL114, whose product is homologous to the uracil DNA glycosylase and is highly conserved in all herpesviruses. This DNA repair enzyme excises uracil residues in DNA that result from the misincorporation of dUTP or spontaneous deamination of cytosine. We constructed a recombinant virus, RC2620, that contains a large deletion in the UL114 open reading frame and carries a 1.2-kb insert containing the Escherichia coli gpt gene. RC2620 retains the capacity to replicate in primary human fibroblasts and reaches titers that are similar to those produced by the parent virus but exhibits a significantly longer replication cycle. Although the rate of expression of alpha and beta gene products appears to be unaffected by the mutation, DNA synthesis fails to proceed normally. Once initiated, DNA synthesis in mutant virus-infected cells proceeds at the same rate as with wild-type virus, but initiation is delayed by 48 h. The mutant virus also exhibits two predicted phenotypes: (i) hypersensitivity to the nucleoside analog 5-bromodeoxyuridine and (ii) retention of more uracil residues in genomic DNA than the parental virus. Together, these data suggest UL114 is required for the proper excision of uracil residues from viral DNA but in addition plays some role in establishing the correct temporal progression of DNA synthesis and viral replication. Although such involvement has not been previously observed in herpesviruses, a requirement for uracil DNA glycosylase in DNA replication has been observed in poxviruses.     

Replication fork collapse is a major cause of the high mutation frequency at three-base lesion clusters

Unresolved repair of clustered DNA lesions can lead to the formation of deleterious double strand breaks (DSB) or to mutation induction. Here, we investigated the outcome of clusters composed of base lesions for which base excision repair enzymes have different kinetics of excision/incision. We designed multiply damaged sites (MDS) composed of a rapidly excised uracil (U) and two oxidized bases, 5-hydroxyuracil (hU) and 8-oxoguanine (oG), excised more slowly. Plasmids harboring these U-oG/hU MDS-carrying duplexes were introduced into Escherichia coli cells either wild type or deficient for DNA n-glycosylases. Induction of DSB was estimated from plasmid survival and mutagenesis determined by sequencing of surviving clones. We show that a large majority of MDS is converted to DSB, whereas almost all surviving clones are mutated at hU. We demonstrate that mutagenesis at hU is correlated with excision of the U placed on the opposite strand. We propose that excision of U by Ung initiates the loss of U-oG-carrying strand, resulting in enhanced mutagenesis at the lesion present on the opposite strand. Our results highlight the importance of the kinetics of excision by base excision repair DNA n-glycosylases in the processing and fate of MDS and provide evidence for the role of strand loss/replication fork collapse during the processing of MDS on their mutational consequences.