DNA-uracil and human pathology

Uracil is usually an inappropriate base in DNA, but it is also a normal intermediate during somatic hypermutation (SHM) and class switch recombination (CSR) in adaptive immunity. In addition, uracil is introduced into retroviral DNA by the host as part of a defence mechanism. The sources of uracil in DNA are spontaneous or enzymatic deamination of cytosine (U:G mispairs) and incorporation of dUTP (U:A pairs). Uracil in DNA is removed by a uracil-DNA glycosylase. The major ones are nuclear UNG2 and mitochondrial UNG1 encoded by the UNG-gene, and SMUG1 that also removes oxidized pyrimidines, e.g. 5-hydroxymethyluracil. The other ones are TDG that removes U and T from mismatches, and MBD4 that removes U from CpG contexts. UNG2 is found in replication foci during the S-phase and has a distinct role in repair of U:A pairs, but it is also important in U:G repair, a function shared with SMUG1. SHM is initiated by activation-induced cytosine deaminase (AID), followed by removal of U by UNG2. Humans lacking UNG2 suffer from recurrent infections and lymphoid hyperplasia, and have skewed SHM and defective CSR, resulting in elevated IgM and strongly reduced IgG, IgA and IgE. UNG-defective mice also develop B-cell lymphoma late in life. In the defence against retrovirus, e.g. HIV-1, high concentrations of dUTP in the target cells promotes misincorporation of dUMP-, and host cell APOBEC proteins may promote deamination of cytosine in the viral DNA. This facilitates degradation of viral DNA by UNG2 and AP-endonuclease. However, viral proteins Vif and Vpr counteract this defense by mechanisms that are now being revealed. In conclusion, uracil in DNA is both a mutagenic burden and a tool to modify DNA for diversity or degradation.     

Latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus recruits uracil DNA glycosylase 2 at the terminal repeats and is important for latent persistence of the virus

Latency-associated nuclear antigen (LANA) of KSHV is expressed in all forms of Kaposi's sarcoma-associated herpesvirus (KSHV)-mediated tumors and is important for TR-mediated replication and persistence of the virus. LANA does not exhibit any enzymatic activity by itself but is critical for replication and maintenance of the viral genome. To identify LANA binding proteins, we used a LANA binding sequence 1 DNA affinity column and determined the identities of a number of proteins associated with LANA. One of the identified proteins was uracil DNA glycosylase 2 (UNG2). UNG2 is important for removing uracil residues yielded after either misincorporation of dUTP during replication or deamination of cytosine. The specificity of the 'LANA-UNG2 interaction was confirmed by using a scrambled DNA sequence affinity column. Interaction of LANA and UNG2 was further confirmed by in vitro binding and coimmunoprecipitation assays. Colocalization of these proteins was also detected in primary effusion lymphoma (PEL) cells, as well as in a cotransfected KSHV-negative cell line. UNG2 binds to the carboxyl terminus of LANA and retains its enzymatic activity in the complex. However, no major effect on TR-mediated DNA replication was observed when a UNG2-deficient (UNG(-/-)) cell line was used. Infection of UNG(-/-) and wild-type mouse embryonic fibroblasts with KSHV did not reveal any difference; however, UNG(-/-) cells produced a significantly reduced number of virion particles after induction. Interestingly, depletion of UNG2 in PEL cells with short hairpin RNA reduced the number of viral genome copies and produced infection-deficient virus.     

Activity profiles of enzymes that control the uracil incorporation into DNA during neuronal development.

We have shown that DNA polymerase beta, the only nuclear DNA polymerase present in adult neurons, cannot discriminate between dTTP and dUTP, having the same Km for both substrates. This fact suggests that during reparative DNA synthesis, in adult neurons, dUMP residues can be incorporated into DNA. Since uracil DNA-glycosylase functions to prevent the mutagenic effects of uracil in DNA coming as a product of deamination of cytosine residues or as a result of dUMP incorporation by DNA polymerase, we have studied the perinatal activity of uracil DNA-glycosylase and of 2 enzymes (nucleoside diphosphokinase and dUTPase) involved in dUTP metabolism. Our data indicate that during neuronal development there is a rapid decrease in uracil DNA-glycosylase which could impair the removal of uracil present in DNA in adult neurons. However, misincorporation of dUMP into DNA might be kept to a low frequency by the action of dUTPase present at all developmental stages.     

Reversion of the lethal phenotype of an HIV-1 integrase mutant virus by overexpression of the same integrase mutant protein

The human immunodeficiency virus type 1 (HIV-1) integrase (IN) is essential for integration of viral DNA into host cell chromatin. We have reported previously (Priet, S., Navarro, J. M., Gros, N., Querat, G., and Sire, J. (2003) J. Biol. Chem. 278, 4566-4571) that IN also plays a role in the packaging of the host uracil DNA glycosylase UNG2 into viral particles and that the region of IN encompassing residues 170-180 was responsible for the interaction with UNG2 and for its packaging into virions. In this work, we aimed to investigate the replication of HIV-1 viruses rendered deficient in virion-associated UNG2 by single or double point mutations in the region 170-180 of IN. We show that the L172A/K173A IN mutant virus was deficient for UNG2 packaging and was defective for replication because of a blockage at the stage of proviral DNA integration in host cell DNA. In vitro assays using long term repeat mimics, however, demonstrate that the L172A/K173A IN mutant was catalytically active. Moreover, trans-complementation experiments show that the viral propagation of L172A/K173A viruses could be rescued by the overexpression of Vpr.L172A/K173A IN fusion protein in a dose-dependent manner and that this rescue is independent of UNG2 packaging. Altogether, our data indicate that L172A/K173A mutations of IN induce a subtle defect in the function of IN, which nevertheless dramatically impairs viral replication. Unexpectedly, this blockage of replication could be overcome by forcing the packaging of higher amounts of this same mutated integrase. This is the first study reporting that blockage of the integration process of HIV-1 provirus carrying a mutation of IN could be alleviated by increasing amounts of IN even carrying the same mutations.     

New insights on the role of the gamma-herpesvirus uracil-DNA glycosylase leucine loop revealed by the structure of the Epstein-Barr virus enzyme in complex with an inhibitor protein

Epstein-Barr virus (EBV) is a human gamma-herpesvirus. Within its 86 open reading frame containing genome, two enzymes avoiding uracil incorporation into DNA can be found: uracil triphosphate hydrolase and uracil-DNA glycosylase (UNG). The latter one excises uracil bases that are due to cytosine deamination or uracil misincorporation from double-stranded DNA substrates. The EBV enzyme belongs to family 1 UNGs. We solved the three-dimensional structure of EBV UNG in complex with the uracil-DNA glycosylase inhibitor protein (Ugi) from bacteriophage PBS-2 at a resolution of 2.3 A by X-ray crystallography. The structure of EBV UNG encoded by the BKRF3 reading frame shows the excellent global structural conservation within the solved examples of family 1 enzymes. Four out of the five catalytic motifs are completely conserved, whereas the fifth one, the leucine loop, carries a seven residue insertion. Despite this insertion, catalytic constants of EBV UNG are similar to those of other UNGs. Modelling of the EBV UNG-DNA complex shows that the longer leucine loop still contacts DNA and is likely to fulfil its role of DNA binding and deformation differently than the enzymes with previously solved structures. We could show that despite the evolutionary distance of EBV UNG from the natural host protein, bacteriophage Ugi binds with an inhibitory constant of 8 nM to UNG. This is due to an excellent specificity of Ugi for conserved elements of UNG, four of them corresponding to catalytic motifs and a fifth one corresponding to an important beta-turn structuring the catalytic site.     

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.     

Uracil-DNA glycosylase (UNG)-deficient mice reveal a primary role of the enzyme during DNA replication

Gene-targeted knockout mice have been generated lacking the major uracil-DNA glycosylase, UNG. In contrast to ung- mutants of bacteria and yeast, such mice do not exhibit a greatly increased spontaneous mutation frequency. However, there is only slow removal of uracil from misincorporated dUMP in isolated ung-/- nuclei and an elevated steady-state level of uracil in DNA in dividing ung-/- cells. A backup uracil-excising activity in tissue extracts from ung null mice, with properties indistinguishable from the mammalian SMUG1 DNA glycosylase, may account for the repair of premutagenic U:G mispairs resulting from cytosine deamination in vivo. The nuclear UNG protein has apparently evolved a specialized role in mammalian cells counteracting U:A base pairs formed by use of dUTP during DNA synthesis.     

Structure and specificity of the vertebrate anti-mutator uracil-DNA glycosylase SMUG1

Cytosine deamination is a major promutagenic process, generating G:U mismatches that can cause transition mutations if not repaired. Uracil is also introduced into DNA via nonmutagenic incorporation of dUTP during replication. In bacteria, uracil is excised by uracil-DNA glycosylases (UDG) related to E. coli UNG, and UNG homologs are found in mammals and viruses. Ung knockout mice display no increase in mutation frequency due to a second UDG activity, SMUG1, which is specialized for antimutational uracil excision in mammalian cells. Remarkably, SMUG1 also excises the oxidation-damage product 5-hydroxymethyluracil (HmU), but like UNG is inactive against thymine (5-methyluracil), a chemical substructure of HmU. We have solved the crystal structure of SMUG1 complexed with DNA and base-excision products. This structure indicates a more invasive interaction with dsDNA than observed with other UDGs and reveals an elegant water displacement/replacement mechanism that allows SMUG1 to exclude thymine from its active site while accepting HmU.     

Origin of endogenous DNA abasic sites in Saccharomyces cerevisiae

Abasic (AP) sites are among the most frequent endogenous lesions in DNA and present a strong block to replication. In Saccharomyces cerevisiae, an apn1 apn2 rad1 triple mutant is inviable because of its incapacity to repair AP sites and related 3'-blocked single-strand breaks (M. Guillet and S. Boiteux, EMBO J. 21:2833, 2002). Here, we investigated the origin of endogenous AP sites in yeast. Our results show that the deletion of the UNG1 gene encoding the uracil DNA glycosylase suppresses the lethality of the apn1 apn2 rad1 mutant. In contrast, inactivation of the MAG1, OGG1, or NTG1 and NTG2 genes encoding DNA glycosylases involved in the repair of alkylation or oxidation damages does not suppress lethality. Although viable, the apn1 apn2 rad1 ung1 mutant presents growth delay due to a G(2)/M checkpoint. These results point to uracil as a critical source of the formation of endogenous AP sites in DNA. Uracil can arise in DNA by cytosine deamination or by the incorporation of dUMP during replication. Here, we show that the overexpression of the DUT1 gene encoding the dUTP pyrophosphatase (Dut1) suppresses the lethality of the apn1 apn2 rad1 mutant. Therefore, this result points to the dUTP pool as an important source of the formation of endogenous AP sites in eukaryotes.     

Defective Repair of Uracil Causes Telomere Defects in Mouse Hematopoietic Cells

Uracil in the genome can result from misincorporation of dUTP instead of dTTP during DNA synthesis, and is primarily removed by uracil DNA glycosylase (UNG) during base excision repair. Telomeres contain long arrays of TTAGGG repeats and may be susceptible to uracil misincorporation. Using model telomeric DNA substrates, we showed that the position and number of uracil substitutions of thymine in telomeric DNA decreased recognition by the telomere single-strand binding protein, POT1. In primary mouse hematopoietic cells, uracil was detectable at telomeres, and UNG deficiency further increased uracil loads and led to abnormal telomere lengthening. In UNG-deficient cells, the frequencies of sister chromatid exchange and fragility in telomeres also significantly increased in the absence of telomerase. Thus, accumulation of uracil and/or UNG deficiency interferes with telomere maintenance, thereby underscoring the necessity of UNG-initiated base excision repair for the preservation of telomere integrity.     

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.     

DNA Repair Enzyme Uracil DNA Glycosylase Is Specifically Incorporated into Human Immunodeficiency Virus Type 1 Viral Particles through a Vpr-Independent Mechanism

The Vpr protein, encoded by the human immunodeficiency virus type 1 (HIV-1) genome, is one of the nonstructural proteins packaged in large amounts into viral particles. We have previously reported that Vpr associates with the DNA repair enzyme uracil DNA glycosylase (UDG). In this study, we extended these observations by investigating whether UDG is incorporated into virions and whether this incorporation requires the presence of Vpr. Our results, with highly purified viruses, show that UDG is efficiently incorporated either into wild-type virions or into Vpr-deficient HIV-1 virions, indicating that Vpr is not involved in UDG packaging. Using an in vitro protein-protein binding assay, we reveal a direct interaction between the precursor form of UDG and the viral integrase (IN). Finally, we demonstrate that IN-defective viruses fail to incorporate UDG, indicating that IN is required for packaging of UDG into virions.