Efficiency of excision of 8-oxo-guanine within DNA clustered damage by XRS5 nuclear extracts and purified human OGG1 protein.

A major DNA lesion is the strongly mutagenic 8-oxo-7,8-dihydroguanine (8-oxoG) base, formed by oxidative attack at guanine and which leads to a high level of G.C-->T.A transversions. Clustered DNA damages are formed in DNA following exposure to ionizing radiation or radiomimetic anticancer agents and are thought to be biologically severe. The presence of 8-oxoG within clustered DNA damage may present a challenge to the repair machinery of the cell, if the OGG1 DNA glycosylase/AP lyase protein, present in eukaryotic cells, does not efficiently excise its substrate, 8-oxoG. In this study, specific oligonucleotide constructs containing an 8-oxoG located in several positions opposite to another damage (5,6-dihydrothymine (DHT), uracil, 8-oxoG, AP site, or various types of single strand breaks) were used to determine the relative efficiency of purified human OGG1 and mammalian XRS5 nuclear extracts to excise 8-oxoG from clustered damages. A base damage (DHT, uracil, and 8-oxoG) on the opposite strand has little or no influence on the rate of excision of 8-oxoG whereas the presence of either an AP site or various types of single strand breaks has a strong inhibitory effect on the formation of a SSB due to the excision of 8-oxoG by both hOGG1 and the nuclear extract. The binding of hOGG1 to 8-oxoG is not significantly affected by the presence of a neighboring lesion.

Recognition and kinetics for excision of a base lesion within clustered DNA damage by the Escherichia coli proteins Fpg and Nth.

Ionizing radiation and radiomimetic anticancer agents induce clustered DNA damages that are thought to lead to deleterious biological consequences, due to the challenge that clustered damage may present to the repair machinery of the cell. Specific oligonucleotides, containing either dihydrothymine (DHT) or 7,8-dihydro-8-oxoguanine (8-oxoG) opposite to specific lesions at defined positions on the complementary strand, have been used to determine the kinetic constants, K(M), k(cat), and specificity constants, for excision of DHT and 8-oxoG by the Escherichia coli base excision repair proteins, endonuclease III (Nth) and formamidopyrimidine glycosylase (Fpg), respectively. For excision of DHT opposite to 8-oxoadenine by Nth or Fpg proteins, or 8-oxoG opposite to 8-oxoG by Fpg, the major change in the specificity constant occurs when the second lesion on the complementary strand is one base to the site opposite to DHT or 8-oxoG. The specificity constants for excision of DHT or 8-oxoG by both proteins are reduced by up to 2 orders of magnitude when an abasic site or a strand break is opposite on the complementary strand. Whereas the values of K(M) are only slightly affected by the presence of a second lesion, the major change is seen as a reduction in the values of k(cat). The binding of Fpg protein to oligonucleotides containing 8-oxoG is inhibited, particularly when a single strand break is near to 8-oxoG on the complementary strand. It is inferred that not only the binding affinity of Fpg protein to the base lesion but also the rate of excision of the damaged base is reduced by the presence of another lesion, particularly a single strand break or an AP site on the complementary strand.

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.

Clustered DNA damage, influence on damage excision by XRS5 nuclear extracts and Escherichia coli Nth and Fpg proteins.

Ionizing radiation and radiomimetic anticancer agents induce clustered DNA damage, which are thought to reflect the biological severity. Escherichia coli Nth and Fpg and nuclear extracts from XRS5, a Chinese hamster ovary Ku-deficient cell line, have been used to study the influence on their substrate recognition by the presence of a neighboring damage or an abasic site on the opposite strand, as models of clustered DNA damage. These proteins were tested for their efficiency to induce a single-strand break on a (32)P-labeled oligonucleotide containing either an abasic (AP) site, dihydrothymine (DHT), 7,8-dihydro-8-oxo-2'deoxyguanine, or 7, 8-dihydro-8-oxo-2'deoxyadenine at positions 1, 3, or 5 base pairs 5' or 3' to either an AP site or DHT on the labeled strand. DHT excision is much more affected than cleavage of an AP site by the presence of other damage. The effect on DHT excision is greatest with a neighboring AP site, with the effect being asymmetric with Nth and Fpg. Therefore, this large inhibition of the excision of DHT by the presence of an opposite AP site may minimize the formation of double-strand breaks in the processing of DNA clustered damages.

c-Myb protein binds to the EP element of the HBV enhancer and regulates transcription in synergy with NF-M.

The hepatitis B virus (HBV) enhancer contains multiple active elements, one of which is the EP element, a 15 bp site important for its regulation by acting on other functional elements like the E site. The EP element, in the HBV enhancer context, contains two putative binding sites for c-myb family gene products. Electrophoretic mobility shift assays showed that the minimal c-Myb DNA-binding domain binds to the EP sequence. DNase I footprinting experiments revealed that only one consensus binding site was effectively protected. We found that c-Myb down-regulates transcription driving by the HBV enhancer in CAT assays performed in a haematopoietic (K562) and in a hepatic (HepG2) cell line. Interestingly, co-expression of both c-Myb and NF-M, a C/EBPbeta homologue which recognises the E element of the HBV enhancer, showed a synergistic transactivation of the HBV enhancer while, separately, each of them had an inhibitory effect on transcription in HepG2 and K562 cell lines, two cell types potentially infected by the hepatitis B virus.

The DNA-binding domain of human c-Abl tyrosine kinase promotes the interaction of a HMG chromosomal protein with DNA.

The biological activity of the c-Abl protein is linked to its tyrosine kinase and DNA-binding activities. The protein, which plays a major role in the cell cycle response to DNA damage, interacts preferentially with sequences containing an AAC motif and exhibits a higher affinity for bent or bendable DNA, as is the case with high mobility group (HMG) proteins. We have compared the DNA-binding characteristics of the DNA-binding domain of human c-Abl and the HMG-D protein from Drosophila melanogaster. c-Abl binds tightly to circular DNA molecules and potentiates the interaction of DNA with HMG-D. In addition, we used a series of DNA molecules containing modified bases to determine how the exocyclic groups of DNA influence the binding of the two proteins. Interfering with the 2-amino group of purines affects the binding of the two proteins similarly. Adding a 2-amino group to adenines restricts the access of the proteins to the minor groove, whereas deleting this bulky substituent from guanines facilitates the protein-DNA interaction. In contrast, c-Abl and HMG-D respond very differently to deletion or addition of the 5-methyl group of pyrimidine bases in the major groove. Adding a methyl group to cytosines favours the binding of c-Abl to DNA but inhibits the binding of HMG-D. Conversely, deleting the methyl group from thymines promotes the interaction of the DNA with HMG-D but diminishes its interaction with c-Abl. The enhanced binding of c-Abl to DNA containing 5-methylcytosine residues may result from an increased propensity of the double helix to denature locally coupled with a protein-induced reduction in the base stacking interaction. The results show that c-Abl has unique DNA-binding properties, quite different from those of HMG-D, and suggest an additional role for the protein kinase.

The DNA binding domain of the human c-Abl tyrosine kinase preferentially binds to DNA sequences containing an AAC motif and to distorted DNA structures.

The c-Abl tyrosine kinase protein is implicated in the signaling pathway as well as in transcription, DNA repair, apoptosis, and several other vital biological processes essential for cell proliferation or differentiation. The interaction of c-Abl with DNA is important for some of these functions, but the exact nature of this interaction is still a matter of controversy. The present study addresses the DNA-binding properties of the human c-Abl protein. Using CASTing experiments, the consensus binding site 5'-AA/CAACAAA/C was determined. The central highly conserved AAC triplet appears to constitute the crucial core element in the binding sequences of the c-Abl protein. The c-Abl DNA-binding domain recognizes specific sequences and interacts with deformed DNA structures such as four-way junctions and bubble DNA containing a large single-stranded loop, as determined by electromobility shift assay, melting temperature studies, and binding to specific oligonucleotides covalently linked to beads. Additional competition experiments suggest that the interaction mainly involves contacts within the minor groove of the double helix. The DNA-binding properties of c-Abl are reminiscent of those of high-mobility group (HMG)-like proteins such as LEF-1 and SRY. However, the circular permutation and ring closure assays and DNA unwinding experiments reveal that, unlike HMGs, c-Abl does not bend its target sequence. In addition, it is shown that the protein potentiates the DNA relaxation activity of topoisomerase I. These findings indicate that the interaction of c-Abl with DNA is both sequence-selective and structure-dependent.

Determination of the human c-Abl consensus DNA binding site.

c-Abl tyrosine kinase, an essential protein of the cell cycle signalling pathways, is implicated in the regulation of RNA polymerase II activity, apoptosis and DNA repair. Its DNA binding activity is important for its biological functions. However, the molecular basis of c-Abl interaction with DNA remains largely unclear. We delimited the human c-Abl DNA binding domain and identified its preferred binding site, 5'-A(A/C)AACAA(A/C). The central AAC motif is highly conserved and constitutes the major core element in the binding sites. EMSAs and footprinting experiments were performed to explore how the c-Abl fusion protein recognizes specific sequences in DNA.

Activation of p65 NF-kappaB protein by p210BCR-ABL in a myeloid cell line (P210BCR-ABL activates p65 NF-kappaB).

The chimeric tyrosine kinase p210BCR-ABL is involved in the pathogenesis of chronic myelogenous leukemia. It transforms immature hematopoietic cells in vitro and abrogates IL-3-dependent growth. The mechanisms by which p210BCR-ABL mediates its oncogenicity are not well elucidated. Identifying transcription factors targeted by the chimeric protein may help to clarify these mechanisms. We have analysed the effect of p210BCR-ABL expression on NF-kappaB activity in DA1 cells (an IL-3-dependent murine myeloid progenitor cell line). A specific stimulation of NF-kappaB activity by kinase-active wild-type p210BCR-ABL has been evidenced by transcriptional activation assays. Electrophoretic mobility supershift assays revealed the presence of p65 protein (RelA) DNA binding activity in p210BCR-ABL transformed DA1 cells but not in parental DA1 cells. Activation of RelA in transformed DA1 cells may occur by protein stabilization. Experiments using oligonucleotides antisense to RelA showed that p210BCR-ABL transfected cells failed to survive after IL-3 removal. Moreover, inhibition of cellular growth was shown following treatment of p210BCR-ABL transformed DA1 cells by p65 antisense oligonucleotides. This study suggests that p65 NF-kappaB may be an effector for p210BCR-ABL and probably contributes to its induced transformation process.