Isolation of a formamidopyrimidine-DNA glycosylase (fpg) mutant of Escherichia coli K12

Summary The fpg ⁺ gene of Escherichia coli coding for formamidopyrimidine-DNA glycosylase was previously cloned on a multicopy plasmid. The plasmid copy of the fpg ⁺ gene was inactivated by cloning a kanamycin resistance gene into the open reading frame, yielding the fpg-1:: Kn^r mutation. This mutation was transferred to the chromosome in the following steps: (i) linearization of the plasmid bearing the fpg-1::Kn^r mutation and transformation of competent bacteria (recB recC sbcB); (ii) selection for chromosomal integration of the fpg-1::Kn^r mutation; (iii) phage P1 mediated transduction of the fpg-1::Kn^r mutation in the AB1157 background. The resulting fpg ^- mutant exhibited no detectable Fapy-DNA glycosylase activity in crude lysates. The insertion mutation was localized by means of genetic crosses between mtl and pyrE, at 81.7 min on the E. coli linkage map. Sequence analysis confirmed this mapping and further showed that fpg is adjacent to rpmBG in the order fpg, rpmGB, pyrE. The formamidopyrimidine-DNA glycosylase defective strain does not show unusual sensitivity to the following DNA damaging treatments: (i) methylmethanesulfonate, (ii) N-methyl-N-nitro-N-nitrosoguanidine, (iii) ultraviolet light, (iv) -radiation. The fpg gene is neither part of the SOS regulon nor the adaptive response to alkylating agents.     

Interaction between APC and Fen1 during breast carcinogenesis

Aberrant DNA base excision repair (BER) contributes to malignant transformation. However, inter-individual variations in DNA repair capacity plays a key role in modifying breast cancer risk. We review here emerging evidence that two proteins involved in BER – adenomatous polyposis coli (APC) and flap endonuclease 1 (Fen1) – promote the development of breast cancer through novel mechanisms. APC and Fen1 expression and interaction is increased in breast tumors versus normal cells, APC interacts with and blocks Fen1 activity in Pol-β-directed LP-BER, and abrogation of LP-BER is linked with cigarette smoke condensate-induced transformation of normal breast epithelial cells. Carcinogens increase expression of APC and Fen1 in spontaneously immortalized human breast epithelial cells, human colon cancer cells, and mouse embryonic fibroblasts. Since APC and Fen1 are tumor suppressors, an increase in their levels could protect against carcinogenesis; however, this does not seem to be the case. Elevated Fen1 levels in breast and lung cancer cells may reflect the enhanced proliferation of cancer cells or increased DNA damage in cancer cells compared to normal cells. Inactivation of the tumor suppressor functions of APC and Fen1 is due to their interaction, which may act as a susceptibility factor for breast cancer. The increased interaction of APC and Fen1 may occur due to polypmorphic and/or mutational variation in these genes. Screening of APC and Fen1 polymorphic and/or mutational variations and APC/Fen1 interaction may permit assessment of individual DNA repair capability and the risk for breast cancer development. Such individuals might lower their breast cancer risk by reducing exposure to carcinogens. Stratifying individuals according to susceptibility would greatly assist epidemiologic studies of the impact of suspected environmental carcinogens. Additionally, a mechanistic understanding of the interaction of APC and Fen1 may provide the basis for developing new and effective targeted chemopreventive and chemotherapeutic agents.     

DNA damage and repair in mammalian cells exposed to p-hydroxyphenylpyruvic acid

Tyrosinemia type 1 (HT1) is an autosomal recessive disorder of the tyrosine metabolism in which the fumarylacetoacetate hydrolase enzyme is defective. This disease is clinically heterogeneous and a chronic and acute form is discerned. Characteristic of the chronic form is the development of cellular hepatocarcinoma. Although p-hydroxyphenylpyruvic acid (pHPPA) is used as one of the diagnostic markers of this disease, it was suggested that it is unlikely to be involved in the pathophysiology of HT1 as it is present in other disorders that does not have hepatorenal symptoms. It was the aim of this study to investigate the possible effect of pHPPA on DNA damage and repair in mammalian cells. The comet assay was used to establish the genotoxicity of pHPPA in human peripheral blood lymphocytes and isolated rat hepatocytes after their exposure to pHPPA. At first glance the damage to DNA caused by pHPPA seemed reparable in both cell types, however, after challenging the DNA repair capacity of metabolite-treated cells with treatment with H(2)O(2), a marked impairment in the DNA repair capability of these cells was observed. We suggest that the main effect of pHPPA is the long-term impairment of the DNA repair machinery rather than the direct damage to DNA and that this effect of pHPPA, together with the other characteristic metabolites, e.g., FAA and MAA, causes cellular hepatocarcinoma to develop in the chronic form of HT1.     

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.     

SUMO-modification and elimination of the active DNA demethylation enzyme TDG in cultured human cells

Thymine DNA glycosylase (TDG) is a base excision repair enzyme that interacts with the small ubiquitin-related modifier (SUMO)-targeted ubiquitin E3 ligase RNF4 and functions in the active DNA demethylation pathway. Here we showed that both SUMOylated and non-modified forms of endogenous TDG fluctuated during the cell cycle and in response to drugs that perturbed cell cycle progression, including hydroxyurea and nocodazole. Additionally, we detected a SUMOylation-independent association between TDG and RNF4 in vitro as well as in vivo, and observed that both forms of TDG were efficiently degraded in RNF4-depleted cells when arrested at S phase. Our findings provide insights into the in vivo dynamics of TDG SUMOylation and further clarify the TDG–RNF4 interaction.     

Viral infection-oxidative stress/DNA damage-aberrant DNA methylation: separate or interrelated events responsible for genetic instability and childhood ALL development?

Acute lymphoblastic leukemia (ALL) is a malignant disorder that originates in a single B- or T-lymphocyte progenitor and is characterized by a range of numeric and structural chromosomal aberrations. Although, so far no clear cause can be found for ALL the most commonly recognized and strongest causal factor is infection. However, an interesting question is how viral infection may be responsible for genetic changes that lead to lymphoid cell transformation. A plausible mechanism by which infection might impact the process of leukemogenesis via genetic alteration is through: oxidative stress/DNA damage which is closely linked with inflammation, aberrant expression of AID/ABOBEC family enzymes which may be responsible for massive mutation introduction and alteration of DNA methylation, leading to changes in the expression of hematopoietic genes. In this review we propose several specific molecular mechanisms which link infection with all the above-mentioned processes. The most likely event which links common virus infection with ALL pathogenesis is aberrant expression of AID/APOBEC. This event may be directly responsible for the introduction of point mutations (as the result of cytosine or 5-methylcytosine deamination and formation of G:U or G:T misspairs) as well as changes in DNA methylation status.     

Lipid peroxidation product 4-hydroxy-2-nonenal modulates base excision repair in human cells

Oxidative-stress-driven lipid peroxidation (LPO) is involved in the pathogenesis of several human diseases, including cancer. LPO products react with cellular proteins changing their properties, and with DNA bases to form mutagenic etheno-DNA adducts, removed from DNA mainly by the base excision repair (BER) pathway.One of the major reactive aldehydes generated by LPO is 4-hydroxy-2-nonenal (HNE). We investigated the effect of HNE on BER enzymes in human cells and in vitro. K21 cells pretreated with physiological HNE concentrations were more sensitive to oxidative and alkylating agents, H₂O₂ and MMS, than were untreated cells. Detailed examination of the effects of HNE on particular stages of BER in K21 cells revealed that HNE decreases the rate of excision of 1,N⁶-ethenoadenine (ɛA) and 3,N⁴-ethenocytosine (ɛC), but not of 8-oxoguanine. Simultaneously HNE increased the rate of AP-site incision and blocked the re-ligation step after the gap-filling by DNA polymerases. This suggested that HNE increases the number of unrepaired single-strand breaks (SSBs) in cells treated with oxidizing or methylating agents. Indeed, preincubation of cells with HNE and their subsequent treatment with H₂O₂ or MMS increased the number of nuclear poly(ADP-ribose) foci, known to appear in cells in response to SSBs. However, when purified BER enzymes were exposed to HNE, only ANPG and TDG glycosylases excising ɛA and ɛC from DNA were inhibited, and only at high HNE concentrations. APE1 endonuclease and 8-oxoG-DNA glycosylase 1 (OGG1) were not inhibited. These results indicate that LPO products exert their promutagenic action not only by forming DNA adducts, but in part also by compromising the BER pathway.