Excision of uracil from DNA by the hyperthermophilic Afung protein is dependent on the opposite base and stimulated by heat-induced transition to a more open structure.

Hydrolytic deamination of DNA-cytosines into uracils is a major source of spontaneously induced mutations, and at elevated temperatures the rate of cytosine deamination is increased. Uracil lesions are repaired by the base excision repair pathway, which is initiated by a specific uracil DNA glycosylase enzyme (UDG). The hyperthermophilic archaeon Archaeoglobus fulgidus contains a recently characterized novel type of UDG (Afung), and in this paper we describe the over-expression of the afung gene and characterization of the encoded protein. Fluorescence and activity measurements following incubation at different temperatures may suggest the following model describing structure-activity relationships: At temperatures from 20 to 50 degrees C Afung exists as a compact protein exhibiting low enzyme activity, whereas at temperatures above 50 degrees C, the Afung conformation opens up, which is associated with the acquisition of high enzyme activity. The enzyme exhibits opposite base-dependent excision of uracil in the following order: U>U:T>U:C>U:G>U:A. Afung is product-inhibited by uracil and shows a pronounced inhibition by p-hydroxymercuribenzoate, indicating a cysteine residue essential for enzyme function. The Afung protein was estimated to be present in A. fulgidus at a concentration of approximately 1000 molecules per cell. Kinetic parameters determined for Afung suggest a significantly lower level of enzymatic uracil release in A. fulgidus as compared to the mesophilic Escherichia coli.     

Uracil DNA glycosylase disruption blocks Ig gene conversion and induces transition mutations

Ig gene conversion is most likely initiated by activation-induced cytidine deaminase-mediated cytosine deamination. If the resulting uracils need to be further processed by uracil DNA glycosylase (UNG), UNG inactivation should block gene conversion and induce transition mutations. In this study, we report that this is indeed the phenotype in the B cell line DT40. Ig gene conversion is almost completely extinguished in the UNG-deficient mutant and large numbers of transition mutations at C/G bases accumulate within the rearranged Ig L chain gene (IgL). The mutation rate of UNG-deficient cells is about seven times higher than that of pseudo V gene-deleted (psiV-) cells in which mutations arise presumably after uracil excision. In addition, UNG-deficient cells show relatively more mutations upstream and downstream of the VJ segment. This suggests that hypermutating B cells process activation-induced cytidine deaminase-induced uracils with approximately one-seventh of uracils giving rise to mutations depending on their position.     

Mapping structurally perturbed sites in DNA by replication arrest and run-off replication

We describe a technique for rapid fine mapping of sites of torsion-induced perturbations of DNA structure. The technique involves strand scission or chemical base modification at structurally perturbed sites, replication arrest in a double-strand DNA sequencing reaction, and size analysis of replication products by electrophoresis on sequencing gels. Besides being less complicated and faster than site identification by conventional end-labeling methods, the technique assures high sequence specificity through the use of oligomeric sequencing primers. This property should be useful for in vivo mapping of DNA structural perturbations with known sequence within complex genomes.     

Identification and resolution of artifacts in bisulfite sequencing

Bisulfite sequencing has become the most widely used application to detect 5-methylcytosine (5-MeC) in DNA, and provides a reliable way of detecting any methylated cytosine at single-molecule resolution in any sequence context. The process of bisulfite treatment exploits the different sensitivity of cytosine and 5-MeC to deamination by bisulfite under acidic conditions, in which cytosine undergoes conversion to uracil while 5-MeC remains unreactive. In this article, we address the more commonly encountered experimental artifacts associated with bisulfite sequencing, and provide methods for the detection and elimination of these artifacts. In particular, we focus on conditions that inhibit complete bisulfite-mediated conversion of cytosines in a target sequence, and demonstrate the necessity of complete protein removal from DNA samples prior to bisulfite treatment. We also include a brief summary of the experimental protocol for bisulfite treatment and tips for designing polymerase chain reaction (PCR) primers to amplify from bisulfite-treated DNA.     

High-Speed Conversion of Cytosine to Uracil in Bisulfite Genomic Sequencing Analysis of DNA Methylation

Bisulfite genomic sequencing is a widely used technique foranalyzing cytosine-methylation of DNA. By treating DNA withbisulfite, cytosine residues are deaminated to uracil, whileleaving 5-methylcytosine largely intact. Subsequent PCR andnucleotide sequence analysis permit unequivocal determinationof the methylation status at cytosine residues. A major caveatassociated with the currently practiced procedure is that ittakes 16–20 hr for completion of the conversion of cytosineto uracil. Here we report that a complete deamination of cytosineto uracil can be achieved in shorter periods by using a highlyconcentrated bisulfite solution at an elevated temperature.Time course experiments demonstrated that treating DNA with9 M bisulfite for 20 min at 90°C or 40 min at 70°C allcytosine residues in the DNA were converted to uracil. Underthese conditions, the majority of 5-methylcytosines remainedintact. When a high molecular weight DNA derived from a cellline (containing a number of genes whose methylation statuswas known) was treated with bisulfite under the above conditionsand amplified and sequenced, the results obtained were consistentwith those reported in the literature. Although some degradationof DNA occurred during this process, the amount of treated DNArequired for the amplification was nearly equal to that requiredfor the conventional bisulfite genomic sequencing procedure.The increased speed of DNA methylation analysis with this novelprocedure is expected to advance various aspects of DNA sciences.     

DNA Methylation: Bisulphite Modification and Analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5''-CpG-3'' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.¹ and Clark et al.², methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.     

Crystal structure of a family 4 uracil-DNA glycosylase from Thermus thermophilus HB8

Uracil-DNA glycosylase (UDG; EC 3.2.2.-) removes uracil from DNA to initiate DNA base excision repair. Since hydrolytic deamination of cytosine to uracil is one of the most frequent DNA-damaging events in all cells, UDG is an essential enzyme for maintaining the integrity of genomic information. For the first time, we report the crystal structure of a family 4 UDG from Thermus thermophilus HB8 (TthUDG) complexed with uracil, solved at 1.5 angstroms resolution. As opposed to UDG enzymes in its other families, TthUDG possesses a [4Fe-4S] cluster. This iron-sulfur cluster, which is distant from the active site, interacts with loop structures and has been suggested to be unessential to the activity but necessary for stabilizing the loop structures. In addition to the iron-sulfur cluster, salt-bridges and ion pairs on the molecular surface and the presence of proline on loops and turns is thought to contribute to the enzyme's thermostability. Despite very low levels of sequence identity with Escherichia coli and human UDGs (family 1) and E.coli G:T/U mismatch-specific DNA glycosylase (MUG) (family 2), the topology and order of secondary structures of TthUDG are similar to those of these distant relatives. Furthermore, the coordinates of the core structure formed by beta-strands are almost the same. Positive charge is distributed over the active-site groove, where TthUDG would bind DNA strands, as do UDG enzymes in other families. TthUDG recognizes uracil specifically in the same manner as does human UDG (family 1), rather than guanine in the complementary strand DNA, as does E.coli MUG (family 2). These results suggest that the mechanism by which family 4 UDGs remove uracils from DNA is similar to that of family 1 enzymes.     

Novel activities of human uracil DNA N-glycosylase for cytosine-derived products of oxidative DNA damage.

Uracil DNA N-glycosylase is a repair enzyme that releases uracil from DNA. A major function of this enzyme is presumably to protect the genome from pre-mutagenic uracil resulting from deamination of cytosine in DNA. Here, we report that human uracil DNA N-glycosylase also recognizes three uracil derivatives that are generated as major products of cytosine in DNA by hydroxyl radical attack or other oxidative processes. DNA substrates were prepared by gamma-irradiation of DNA in aerated aqueous solution and incubated with human uracil DNA N-glycosylase, heat-inactivated enzyme or buffer. Ethanol-precipitated DNA and supernatant fractions were then separated. Supernatant fractions after derivatization, and pellets after hydrolysis and derivatization were analyzed by gas chromatography/isotope-dilution mass spectrometry. The results demonstrated that human uracil DNA N-glycosylase excised isodialuric acid, 5-hydroxyuracil and alloxan from DNA with apparent K(m) values of approximately 530, 450 and 660 nM, respectively. The excision of these uracil analogues is consistent with the recently described mechanism for recognition of uracil by human uracil DNA N-glycosylase [Mol,C.D., Arval,A.S., Slupphaug,G., Kavil,B., Alseth,I., Krokan,H.E. and Tainer,J.A. (1995) Cell, 80, 869-878]. Nine other pyrimidine- and purine-derived products that were identified in DNA samples were not substrates for the enzyme. The results indicate that human uracil DNA N-glycosylase may have a function in the repair of oxidative DNA damage.     

Sequence analysis and editing for bisulphite genomic sequencing projects

Bisulphite genomic sequencing is a widely used technique for detailed analysis of the methylation status of a region of DNA. It relies upon the selective deamination of unmethylated cytosine to uracil after treatment with sodium bisulphite, usually followed by PCR amplification of the chosen target region. Since this two-step procedure replaces all unmethylated cytosine bases with thymine, PCR products derived from unmethylated templates contain only three types of nucleotide, in unequal proportions. This can create a number of technical difficulties (e.g. for some base-calling methods) and impedes manual analysis of sequencing results (since the long runs of T or A residues are difficult to align visually with the parent sequence). To facilitate the detailed analysis of bisulphite PCR products (particularly using multiple cloned templates), we have developed a visually intuitive program that identifies the methylation status of CpG dinucleotides by analysis of raw sequence data files produced by MegaBace or ABI sequencers as well as Staden SCF trace files and plain text files. The program then also collates and presents data derived from independent templates (e.g. separate clones). This results in a considerable reduction in the time required for completion of a detailed genomic methylation project.     

The specific binding of nuclear protein(s) to the cAMP responsive element (CRE) sequence (TGACGTCA) is reduced by the misincorporation of U and increased by the deamination of C.

Point mutations in the cAMP-responsive element (CRE) of the rat somatostatin gene promoter/enhancer sequence (TGACGTCA) were used as a model for assessing the effect of uracil, deriving either from misincorporation during DNA synthesis (T----U) or cytosine deamination (C----U), on the binding of sequence/specific regulatory proteins. The results show that the T----U conversion in both strands of the CRE palindromic sequence reduces its affinity for the CRE binding factor(s), suggesting the crucial role of the methyl group contributed by T for the correct recognition of the sequence. On the other hand, deamination of C in the CpG central dinucleotide (CpG----UpG) causes an increase of binding affinity which is further enhanced by the contemporary deamination in both strands. Then, both uracil misincorporation and cytosine deamination alter the binding to CRE sequence in vitro, suggesting that uracil, if not removed by uracil DNA-glycosylase, could be dangerous for cellular functions.     

A rapid and efficient method for region- and strand-specific mutagenesis of cloned DNA.

The single-stranded viral DNA of an M13 phage recombinant containing the early promoter region of SV40 was hybridized with linear, double-stranded replicative form DNA of a related M13 phage containing a short deletion in the cloned SV40 sequence. The heteroduplexes formed between these DNA molecules contained a short, defined single-stranded region in an otherwise duplex molecule. These heteroduplexes were treated with sodium bisulphite to deaminate exposed unpaired cytosines to uracil residues. The single-stranded region was filled in with DNA polymerase I, which incorporates adenine opposite the mutated uracils, and the DNA then transfected into the M13 host JM103 . Viral DNA from the resultant plaques was used for the rapid dideoxy-DNA sequencing procedure; all of the plaques studied contained point mutations within the desired area. This method allows the very rapid and efficient generation of region-directed point mutants which can be quickly sequenced.     

Enzymology of base excision repair in the hyperthermophilic archaeon Pyrobaculum aerophilum

DNA of all living organisms is constantly modified by exogenous and endogenous reagents. The mutagenic threat of modifications such as methylation, oxidation, and hydrolytic deamination of DNA bases is counteracted by base excision repair (BER). This process is initiated by the action of one of several DNA glycosylases, which removes the aberrant base and thus initiates a cascade of events that involves scission of the DNA backbone, removal of the baseless sugar-phosphate residue, filling in of the resulting single nucleotide gap, and ligation of the remaining nick. We were interested to find out how the BER process functions in hyperthermophiles, organisms growing at temperatures around 100 degrees C, where the rates of these spontaneous reactions are greatly accelerated. In our previous studies, we could show that the crenarchaeon Pyrobaculum aerophilum has at least three uracil-DNA glycosylases, Pa-UDGa, Pa-UDGb, and Pa-MIG, that can initiate the BER process by catalyzing the removal of uracil residues arising through the spontaneous deamination of cytosines. We now report that the genome of P. aerophilum encodes also the remaining functions necessary for BER and show that a system consisting of four P. aerophilum encoded enzymes, Pa-UDGb, AP endonuclease IV, DNA polymerase B2, and DNA ligase, can efficiently repair a G.U mispair in an oligonucleotide substrate to a G.C pair. Interestingly, the efficiency of the in vitro repair reaction was stimulated by Pa-PCNA1, the processivity clamp of DNA polymerases.     

Genomic uracil and human disease

Uracil is present in small amounts in DNA due to spontaneous deamination of cytosine and incorporation of dUMP during replication. While deamination generates mutagenic U:G mismatches, incorporated dUMP results in U:A pairs that are not directly mutagenic, but may be cytotoxic. In most cells, mutations resulting from uracil in DNA are prevented by error-free base excision repair. However, in B-cells uracil in DNA is also a physiological intermediate in acquired immunity. Here, activation-induced cytosine deaminase (AID) introduces template uracils that give GC to AT transition mutations in the Ig locus after replication. When uracil-DNA glycosylase (UNG2) removes uracil, error-prone translesion synthesis over the abasic site causes other mutations in the Ig locus. Together, these processes are central to somatic hypermutation (SHM) that increases immunoglobulin diversity. AID and UNG2 are also essential for generation of strand breaks that initiate class switch recombination (CSR). Patients lacking UNG2 display a hyper-IgM syndrome with recurrent infections, increased IgM, strongly decreased IgG, IgA and IgE and skewed SHM. UNG2 is also involved in innate immune response against retroviral infections. Ung(-/-) mice have a similar phenotype and develop B-cell lymphomas late in life. However, there is no evidence indicating that UNG deficiency causes lymphomas in humans.     

Genomic Uracil Homeostasis during Normal B Cell Maturation and Loss of This Balance during B Cell Cancer Development

Activation-induced deaminase (AID) converts DNA cytosines to uracils in immunoglobulin genes, creating antibody diversification. It also causes mutations and translocations that promote cancer. We examined the interplay between uracil creation by AID and its removal by UNG2 glycosylase in splenocytes undergoing maturation and in B cell cancers. The genomic uracil levels remain unchanged in normal stimulated B cells, demonstrating a balance between uracil generation and removal. In stimulated UNG^−/− cells, uracil levels increase by 11- to 60-fold during the first 3 days. In wild-type B cells, UNG2 gene expression and enzymatic activity rise and fall with AID levels, suggesting that UNG2 expression is coordinated with uracil creation by AID. Remarkably, a murine lymphoma cell line, several human B cell cancer lines, and human B cell tumors expressing AID at high levels have genomic uracils comparable to those seen with stimulated UNG^−/−splenocytes. However, cancer cells express UNG2 gene at levels similar to or higher than those seen with peripheral B cells and have nuclear uracil excision activity comparable to that seen with stimulated wild-type B cells. We propose that more uracils are created during B cell cancer development than are removed from the genome but that the uracil creation/excision balance is restored during establishment of cell lines, fixing the genomic uracil load at high levels.     

Enzymatic excision of uracil residues in nucleosomes depends on the local DNA structure and dynamics

The excision of uracil bases from DNA is accomplished by the enzyme uracil DNA glycosylase (UNG). Recognition of uracil bases in free DNA is facilitated by uracil base pair dynamics, but it is not known whether this same mechanistic feature is relevant for detection and excision of uracil residues embedded in nucleosomes. Here we investigate this question using nucleosome core particles (NCPs) generated from Xenopus laevis histones and the high-affinity "Widom 601" positioning sequence. The reactivity of uracil residues in NCPs under steady-state multiple-turnover conditions was generally decreased compared to that of free 601 DNA, mostly because of anticipated steric effects of histones. However, some sites in NCPs had equal or even greater reactivity than free DNA, and the observed reactivities were not readily explained by simple steric considerations or by global DNA unwrapping models for nucleosome invasion. In particular, some reactive uracils were found in occluded positions, while some unreactive uracils were found in exposed positions. One feature of many exposed reactive sites is a wide DNA minor groove, which allows penetration of a key active site loop of the enzyme. In single-turnover kinetic measurements, multiphasic reaction kinetics were observed for several uracil sites, where each kinetic transient was independent of the UNG concentration. These kinetic measurements, and supporting structural analyses, support a mechanism in which some uracils are transiently exposed to UNG by local, rate-limiting nucleosome conformational dynamics, followed by rapid trapping of the exposed state by the enzyme. We present structural models and plausible reaction mechanisms for the reaction of UNG at three distinct uracil sites in the NCP.     

Thermal resistance to photoreactivation of specific mutations potentiated in E. coli B/r ung by ultraviolet light

Summary Mutagenesis by ultraviolet light was studied in a strain of E. coli ung, which lacks uracil-DNA glycosylase activity. Mutation potentiated by UV in cells already induced by nalidixic acid treatment was still photoreversible suggesting that pyrimidine dimers act directly as premutational photoproducts. Secondly, irradiated cells were held in buffer at 48°C for 0 to 135 min to allow for deamination of cytosines in pyrimidine dimers. The mutation frequencies for class 2 de novo suppressor mutation, for class 2 converted suppressor mutation and for backmutation were individually determined, before and after photoreactivation, as a function of this thermal treatment. Backmutation remained sensitive to photoreactivation throughout the treatment but de novo and converted suppressor mutations rapidly developed resistance to photoreactivation. This resistance was not seen in an ung ⁺ control. A model is proposed to account for the selective resistance based on the hypothesis that class 2 de novo and converted suppressor mutations normally result from UV by GC to AT transitions at T=C dimers. The model describes deamination of the cytosine residues in these dimers to become uracil residues. In consequence, monomerization by photoreactivation in cells that can not repair uracils in DNA no longer reverses mutation and GC to AT transitions are established at the sites of uracils.     

DNA sequence selectivity of guanine-N7 alkylation by nitrogen mustards is preserved in intact cells.

Nitrogen mustard alkylating agents react with isolated DNA in a sequence selective manner, and the substituent attached to the drug reactive group can impose a distinct sequence preference. It is not clear however to what extent the observed DNA sequence preferences are preserved in intact cells. The highly reiterated sequence of human alpha DNA has been used to determine the sites of guanine-N7 alkylation following treatment of cells with three nitrogen mustards, mechlorethamine, uracil mustard and quinacrine mustard, known to react in isolated DNA with distinctly different sequence preferences. Alpha DNA from drug treated cells was extracted, purified, end-labeled, and a 296 base pair, singly end-labelled, fragment isolated. Following the quantitative conversion of alkylation sites to strand breaks the fragments were separated on DNA sequencing gels. Clear differences were observed between the alkylation patterns of the three compounds, and the selectivities were qualitatively similar to those predicted and observed in the same sequence alkylated in vitro. In particular the unique preferences of uracil and quinacrine mustards for 5'-PyGC-3' and 5'-GT/GPu-3' sequences, respectively, were preserved in intact cells suggesting that the pattern of sequence dependent reactivity is not grossly affected by the nuclear milieu.     

Interaction of the family-B DNA polymerase from the archaeon Pyrococcus furiosus with deaminated bases

The interaction of archaeal family B DNA polymerases with deaminated bases has been examined. As determined previously by our group, the polymerase binds tightly to uracil (the deamination product of cytosine), in single-stranded DNA, and stalls replication on encountering this base. DNA polymerisation was also inhibited by the presence of hypoxanthine, the deamination product of adenine. Quantitative binding assays showed that the polymerase bound DNA containing uracil 1.5-4.5-fold more strongly than hypoxanthine and site-directed mutagenesis suggested that the same pocket was used for interaction with both deaminated bases. In contrast the polymerase was insensitive to xanthine, the deamination product of guanine. Traces of uracil and hypoxanthine in DNA can lead to inhibition of the PCR by archaeal DNA polymerases, an important consideration for biotechnology applications. Dual recognition of uracil and hypoxanthine may be facilitated by binding the bases with the glycosidic bond in the anti and syn conformation, respectively.     

Uracil recognition by replicative DNA polymerases is limited to the archaea, not occurring with bacteria and eukarya

Family B DNA polymerases from archaea such as Pyrococcus furiosus, which live at temperatures ~100°C, specifically recognize uracil in DNA templates and stall replication in response to this base. Here it is demonstrated that interaction with uracil is not restricted to hyperthermophilic archaea and that the polymerase from mesophilic Methanosarcina acetivorans shows identical behaviour. The family B DNA polymerases replicate the genomes of archaea, one of the three fundamental domains of life. This publication further shows that the DNA replicating polymerases from the other two domains, bacteria (polymerase III) and eukaryotes (polymerases δ and ε for nuclear DNA and polymerase γ for mitochondrial) are also unable to recognize uracil. Uracil occurs in DNA as a result of deamination of cytosine, either in G:C base-pairs or, more rapidly, in single stranded regions produced, for example, during replication. The resulting G:U mis-pairs/single stranded uracils are promutagenic and, unless repaired, give rise to G:C to A:T transitions in 50% of the progeny. The confinement of uracil recognition to polymerases of the archaeal domain is discussed in terms of the DNA repair pathways necessary for the elimination of uracil.