DNA Damage Recognition Protein Research Articles

Molecular Dynamic Simulations of Cisplatin- and Oxaliplatin-d(GG) Intrastand Cross-links Reveal Differences in their Conformational Dynamics

Mismatch repair proteins, DNA damage-recognition proteins and translesion DNA polymerases discriminate between Pt-GG adducts containing cis-diammine ligands (formed by cisplatin (CP) and carboplatin) and trans-RR-diaminocyclohexane ligands (formed by oxaliplatin (OX)) and this discrimination is thought to be important in determining differences in the efficacy, toxicity and mutagenicity of these platinum anticancer agents. We have postulated that these proteins recognize differences in conformation and/or conformational dynamics of the DNA containing the adducts. We have previously determined the NMR solution structure of OX-DNA, CP-DNA and undamaged duplex DNA in the 5′-d(CCTCAGGCCTCC)-3′ sequence context and have shown the existence of several conformational differences in the vicinity of the Pt-GG adduct. Here we have used molecular dynamics simulations to explore differences in the conformational dynamics between OX-DNA, CP-DNA and undamaged DNA in the same sequence context. Twenty-five 10 ns unrestrained fully solvated molecular dynamics simulations were performed starting from two different DNA conformations using AMBER v8.0. All 25 simulations reached equilibrium within 4 ns, were independent of the starting structure and were in close agreement with previous crystal and NMR structures. Our data show that the cis-diammine (CP) ligand preferentially forms hydrogen bonds on the 5′ side of the Pt-GG adduct, while the trans-RR-diaminocyclohexane (OX) ligand preferentially forms hydrogen bonds on the 3′ side of the adduct. In addition, our data show that these differences in hydrogen bond formation are strongly correlated with differences in conformational dynamics, specifically the fraction of time spent in different DNA conformations in the vicinity of the adduct, for CP- and OX-DNA adducts. We postulate that differential recognition of CP- and OX-GG adducts by mismatch repair proteins, DNA damage-recognition proteins and DNA polymerases may be due, in part, to differences in the fraction of time that the adducts spend in a conformation favorable for protein binding.

RNA polymerase: The most specific damage recognition protein in cellular responses to DNA damage?

DNA damage induces a number of cellular responses in human cells, including DNA repair, transcriptional reprogramming, delay of cell cycle progression, and apoptosis (1). The most common DNA lesions fall into two broad groups: base lesions and single- and double-strand breaks. Both types of lesions are detected by damage sensors that initiate various response reactions. A central question in understanding these responses is the identity of the damage sensor. The study by Derheimer et al. (2) in a recent issue of PNAS, together with previous studies, suggests that RNA polymerase II (RNAP II) stalled at a damaged DNA base may constitute the most specific signal for DNA repair, DNA damage checkpoints, and apoptosis (Fig. 1). We suggest that RNAP II is an ideal damage sensor because it has the highest selectivity of all known DNA damage recognition proteins.

NMR analysis of [ methyl- 13C]methionine UvrB from Bacillus caldotenax reveals UvrB–domain 4 heterodimer formation in solution

UvrB is a central DNA damage recognition protein involved in bacterial nucleotide excision repair. Structural information has been limited by the apparent disorder of the C-terminal domain 4 in crystal structures of intact UvrB; in solution, the isolated domain 4 is found to form a helix–loop–helix dimer. In order to gain insight into the behavior of UvrB in solution, we have performed NMR studies on [ methyl- 13C]methionine-labeled UvrB from Bacillus caldotenax (molecular mass = 75 kDa). The 13 methyl resonances were assigned on the basis of site-directed mutagenesis and domain deletion. Solvent accessibility was assessed based on the relaxation and chemical shift responses of the probe methyl resonances to the stable nitroxide, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL). M632, located at the potential dimer interface of domain 4, provides an ideal probe for UvrB dimerization behavior. The M632 resonance of UvrB is very broad, consistent with some degree of monomer–dimer exchange and/or conformational instability of the exposed dimer interface. Upon addition of unlabeled domain 4 peptide, the M632 resonance of UvrB sharpens and shifts to a position consistent with a UvrB–domain 4 heterodimer. A dissociation constant ( K D) value of 3.3 μM for the binding constant of UvrB with the domain 4 peptide was derived from surface plasmon resonance studies. Due to the flexibility of the domain 3–4 linker, inferred from limited proteolysis data and from the relaxation behavior of linker residue M607, the position of domain 4 is constrained not by the stiffness of the linking segment but by direct interactions with domains 1–3 in UvrB. In summary, UvrB homodimerization is disfavored, while domain 4 homodimerization and UvrB–domain 4 heterodimerization are allowed.

Attenuated Expression of Xeroderma Pigmentosum Group C Is Associated with Critical Events in Human Bladder Cancer Carcinogenesis and Progression

Xeroderma pigmentosum group C (XPC) is an important DNA damage recognition protein that binds to damaged DNA at a very early stage during DNA repair. The XPC protein is also involved in DNA damage-induced cell cycle checkpoint regulation and apoptosis. XPC defects are associated with many types of solid tumors. The mechanism of the XPC protein in cancer progression, however, remains unclear. In this report, we showed the strong correlation between bladder cancer progression and attenuated XPC protein expression using tissues derived from patients with bladder cancer. The results obtained from our immunohistochemical studies further revealed a strong correlation of XPC deficiency, p53 mutation, and the degree of malignancy of bladder tumors. In addition, the results obtained from our studies have also shown that HT1197 bladder cancer cells, which carry a low-level XPC protein, exhibited a decreased DNA repair capability and were resistant to cisplatin treatment. When an XPC gene cDNA-expression vector was stably transfected into the HT1197 cells, however, the cisplatin treatment-induced apoptotic cell death was increased. Increased p53 and p73 responses following cisplatin treatment were also observed in HT1197 cells stably transfected with XPC cDNA. Taken together, these results suggest that XPC deficiency is an important contributing factor in bladder tumor progression and bladder cancer cell drug resistance.

Genes Regulating the Sensitivity of Solid Tumor Cell Lines to Cytotoxic Agents: A Literature Review

In order to review gene alterations associated with drug responses in vitro to identify candidate genes for predictive chemosensitivity testing, we selected from literature genes fulfilling at least one of the following criteria for the definition of 'in vitro chemosensitivity associated gene': (i) alterations of the gene can be identified in human solid tumor cell lines exhibiting drug-induced resistance; (ii) transfection of the gene induces drug resistance; (iii) down-regulation of the gene increases the drug sensitivity. We then performed Medline searches for papers on the association between gene alterations of the selected genes and chemosensitivity of cancer cell lines, using the name of the gene as a keyword. A total of 80 genes were identified, which were categorized according to the protein encoded by them as follows: transporters (n = 15), drug targets (n = 8), target-associated proteins (n = 7), intracellular detoxifiers (n = 7), DNA repair proteins (n = 10), DNA damage recognition proteins (n = 2), cell cycle regulators (n = 6), mitogenic and survival signal regulators (n = 7), transcription factors (n = 4), cell adhesion-mediated drug resistance protein (n = 1), and apoptosis regulators (n = 13). The association between the gene alterations and chemosensitivity of cancer cell lines was evaluated in 50 studies for 35 genes. The genes for which the association above was shown in two or more studies were those encoding the major vault protein, thymidylate synthetase, glutathione S-transferase pi, metallothionein, tumor suppressor p53, and bcl-2. We conclude that a total of 80 in vitro chemosensitivity associated genes identified in the literature are potential candidates for clinical predictive chemosensitivity testing.

The ING1b tumor suppressor facilitates nucleotide excision repair by promoting chromatin accessibility to XPA

ING1b is the most studied ING family protein and perhaps the most ubiquitously and abundantly expressed. This protein is involved in the regulation of various biological functions ranging from senescence, cell cycle arrest, apoptosis, to DNA repair. ING1b is upregulated by UV irradiation and enhances the removal of bulky nucleic acid photoproducts. In this study, we provide evidence that ING1b mediates nucleotide excision repair by facilitating the access to damaged nucleosomal DNA. We demonstrate that ING1b is not recruited to UV-induced DNA lesions but enhances nucleotide excision repair only in XPC-proficient cells, implying an essential role in early steps of the ‘access, repair, restore’ model. We also find that ING1b alters histone acetylation dynamics upon exposure to UV radiation and induces chromatin relaxation in microccocal nuclease digestion assay, revealing that ING1b may allow better access to nucleotide excision repair machinery. More importantly, ING1b associates with chromatin in a UV-inducible manner and facilitates DNA access to nucleotide excision repair factor XPA. Furthermore, depletion of the endogenous ING1b results to the sensitization of cells at S-phase to UV irradiation. Taken together, these observations establish a role of ING1b acting as a chromatin accessibility factor for DNA damage recognition proteins upon genotoxic injury.

Recruitment of DNA damage recognition and repair pathway proteins following near-IR femtosecond laser irradiation of cells

An 800-nm 200-fs laser is used to produce DNA damage in rat kangaroo (PtK1) and human cystic fibrosis pancreatic adenoma carcinoma (CFPAC-1) cells. Immunofluorescence staining for DNA repair factors in irradiated cells displays localization of gammaH2AX, Nbs1, and Rad50 to the site of irradiation 3 to 30 min following laser exposure. It is concluded that the 200-fs near-infrared laser is an excellent source for the production and study of spatially defined regions of DNA damage.

Specific and Efficient Binding of Xeroderma Pigmentosum Complementation Group A to Double-Strand/Single-Strand DNA Junctions with 3‘- and/or 5‘-ssDNA Branches

Human XPA is an important DNA damage recognition protein in nucleotide excision repair (NER). We previously observed that XPA binds to the DNA lesion as a homodimer [Liu, Y., Liu, Y., Yang, Z., Utzat, C., Wang, G., Basu, A. K., and Zou, Y. (2005) Biochemistry 44, 7361-7368]. Herein we report that XPA recognized undamaged DNA double-strand/single-strand (ds-ssDNA) junctions containing ssDNA branches with binding affinity (Kd = 49.1 +/- 5.1 nM) much higher than its ability to bind to DNA damage. The recognized DNA junction structures include the Y-shape junction (with both 3'- and 5'-ssDNA branches), 3'-overhang junction (with a 3'-ssDNA branch), and 5'-overhang junction (with a 5'-ssDNA branch). Using gel filtration chromatography and gel mobility shift assays, we showed that the highly efficient binding appeared to be carried out by the XPA monomer and that the binding was largely independent of RPA. Furthermore, XPA efficiently bound to six-nucleotide mismatched DNA bubble substrates with or without DNA adducts including C8 guanine adducts of AF, AAF, and AP and the T[6,4]T photoproducts. Using a set of defined DNA substrates with varying degrees of DNA bending, we also found that the XPC-HR23B complex recognized DNA bending, whereas neither XPA nor the XPA-RPA complex could bind to bent DNA. We propose that, besides DNA damage recognition, XPA may also play a novel role in stabilizing, via its high affinity to ds-ssDNA junctions, the DNA strand opening surrounding the lesion for stable formation of preincision NER intermediates. Our results provide a plausible mechanistic interpretation for the indispensable requirement of XPA for both global genome and transcription-coupled repairs. Since ds-ssDNA junctions are common intermediates in many DNA metabolic pathways, the additional potential role of XPA in cellular processes is discussed.

UvrB Domain 4, an Autoinhibitory Gate for Regulation of DNA Binding and ATPase Activity

UvrB, a central DNA damage recognition protein in bacterial nucleotide excision repair, has weak affinity for DNA, and its ATPase activity is activated by UvrA and damaged DNA. Regulation of DNA binding and ATP hydrolysis by UvrB is poorly understood. Using atomic force microscopy and biochemical assays, we found that truncation of domain 4 of Bacillus caldotenax UvrB (UvrBDelta4) leads to multiple changes in protein function. Protein dimerization decreases with an approximately 8-fold increase of the equilibrium dissociation constant and an increase in DNA binding. Loss of domain 4 causes the DNA binding mode of UvrB to change from dimer to monomer, and affinity increases with the apparent dissociation constants on nondamaged and damaged single-stranded DNA decreasing 22- and 14-fold, respectively. ATPase activity by UvrBDelta4 increases 14- and 9-fold with and without single-stranded DNA, respectively, and UvrBDelta4 supports UvrA-independent damage-specific incision by Cho on a bubble DNA substrate. We propose that other than its previously discovered role in regulating protein-protein interactions, domain 4 is an autoinhibitory domain regulating the DNA binding and ATPase activities of UvrB.

A novel DNA damage recognition protein in Schizosaccharomyces pombe

Toxic and mutagenic O6-alkylguanine adducts in DNA are repaired by O6-alkylguanine-DNA alkyltransferases (MGMT) by transfer of the alkyl group to a cysteine residue in the active site. Comparisons in silico of prokaryotes and lower eukaryotes reveal the presence of a group of proteins [alkyltransferase-like (ATL) proteins] showing amino acid sequence similarity to MGMT, but where the cysteine at the putative active site is replaced by tryptophan. To examine whether ATL proteins play a role in the biological effects of alkylating agents, we inactivated the gene, referred to as atl1+, in Schizosaccharomyces pombe, an organism that does not possess a functional MGMT homologue. The mutants are substantially more susceptible to the toxic effects of the methylating agents, N-methyl-N-nitrosourea, N-methyl-N′nitro-N-nitrosoguanidine and methyl methanesulfonate and longer chain alkylating agents including N-ethyl-N-nitrosourea, ethyl methanesulfonate, N-propyl-N-nitrosourea and N-butyl-N-nitrosourea. Purified Atl1 protein does not transfer methyl groups from O6-methylguanine in [3H]-methylated DNA but reversibly inhibits methyl transfer by human MGMT. Atl1 binds to short single-stranded oligonucleotides containing O6-methyl, -benzyl, -4-bromothenyl or -hydroxyethyl-guanine but does not remove the alkyl group or base and does not cleave the oligonucleotide in the region of the lesion. This suggests that Atl1 acts by binding to O6-alkylguanine lesions and signalling them for processing by other DNA repair pathways. This is the first report describing an activity that protects S.pombe against the toxic effects of O6-alkylguanine adducts and the biological function of a family of proteins that is widely found in prokaryotes and lower eukaryotes.

Differentiating human keratinocytes are deficient in p53 but retain global nucleotide excision repair following ultraviolet radiation

Terminally differentiating keratinocytes constitute the predominant cell type within the skin epidermis and play an important role in the overall photobiology of human skin following ultraviolet radiation. However, the DNA repair capacity of differentiating keratinocytes is unclear, and little is known regarding how such repair activity is regulated in these cells. We systematically compared the global genomic nucleotide excision repair response of cultured undifferentiated human keratinocytes to those that were allowed to differentiate in 1.2 mM Ca(2+), in some cases supplemented with phorbol ester or Vitamin C. Differentiated cells ceased replication and expressed typical markers of differentiation. Following ultraviolet radiation, keratinocytes that were differentiated up to 12 days removed cyclobutane pyrimidine dimers and pyrimidine(6,4)pyrimidone photoproducts from the global genome as efficiently as undifferentiated cells. However, following the onset of calcium-induced differentiation, basal levels of p53 were nearly undetectable by 12 days of differentiation when global repair activity was unaffected. Following ultraviolet radiation, induction of p53 following ultraviolet radiation was abrogated by 6 days of calcium-induced differentiation. Basal levels of mRNA encoding the DNA damage recognition proteins, XPC and DDB2, were relatively insensitive to differentiation and p53 levels. However, following ultraviolet radiation, inductions of mRNA encoding the DNA damage recognition proteins, DDB2 and XPC, were differentially affected by differentiation. Rapid loss of DDB2 mRNA induction was associated with differentiation, while XPC mRNA induction diminished more slowly with differentiation. These results indicate that human keratinocytes preserve global nucleotide excision repair as well as expression of genes encoding key DNA damage recognition proteins well into the terminal differentiation process, perhaps using mechanisms other than p53.

HHR23A, a human homolog of Saccharomyces cerevisiae Rad23, regulates xeroderma pigmentosum C protein and is required for nucleotide excision repair

HHR23A and hHR23B are the human homologs of Saccharomyces cerevisiae Rad23. hHR23B is associated with the nucleotide excision repair (NER) factor xeroderma pigmentosum C (XPC) protein and is required for global genome repair. The function of hHR23A is not yet clear. In this study, the potential function of the hHR23A protein was investigated using RNA interference techniques. The hHR23A knock-down (KD) construct diminished the RNA level of hHR23A protein by approximately 60%, and it did not interfere with expression of the hHR23B gene. Based on Southwestern immunoblot and host-cell reactivation assays, hHR23A KD cells were found to be deficient in DNA repair activity against the DNA damage caused by UVC irradiation. In these hHR23A KD cells, the XPC gene was not normally induced by UVC irradiation, indicating that the hHR23A protein is involved in NER through regulation of the DNA damage recognition protein XPC. Co-immunoprecipitation experiments revealed that hHR23A was associated with a small portion of hHR23B and the majority of p53 protein, indicating that hHR23A regulates the function of XPC by its association with the NER activator p53.

The involvement of XPC protein in the cisplatin DNA damaging treatment-mediated cellular response.

Recognition of DNA damage is a critical step for DNA damage-mediated cellular response. XPC is an important DNA damage recognition protein involved in nucleotide excision repair (NER). We have studied the XPC protein in cisplatin DNA damaging treatment-mediated cellular response. Comparison of the microarray data from both normal and XPC-defective human fibroblasts identified 861 XPC-responsive genes in the cisplatin treatment (with minimum fold change > or = 1.5). The cell cycle and cell proliferation-related genes are the most affected genes by the XPC defect in the treatment. Many other cellular function genes, especially the DNA repair and signal transduction-related genes, were also affected by the XPC defect in the treatment. To validate the microarray data, the transcription levels of some microarray-identified genes were also determined by an RT-PCR based real time PCR assay. The real time PCR results are consistent with the microarray data for most of the tested genes, indicating the reliability of the microarray data. To further validate the microarray data, the cisplatin treatment-mediated caspase-3 activation was also determined. The Western blot hybridization results indicate that the XPC defect greatly attenuates the cisplatin treatment-mediated Caspase-3 activation. We elucidated the role of p53 protein in the XPC protein DNA damage recognition-mediated signaling process. The XPC defect reduces the cisplatin treatment-mediated p53 response. These results suggest that the XPC protein plays an important role in the cisplatin treatment-mediated cellular response. It may also suggest a possible mechanism of cancer cell drug resistance.

The NEF4 complex regulates Rad4 levels and utilizes Snf2/Swi2-related ATPase activity for nucleotide excision repair.

Nucleotide excision repair factor 4 (NEF4) is required for repair of nontranscribed DNA in Saccharomyces cerevisiae. Rad7 and the Snf2/Swi2-related ATPase Rad16 are NEF4 subunits. We report previously unrecognized similarity between Rad7 and F-box proteins. Rad16 contains a RING domain embedded within its ATPase domain, and the presence of these motifs in NEF4 suggested that NEF4 functions as both an ATPase and an E3 ubiquitin ligase. Mutational analysis provides strong support for this model. The Rad16 ATPase is important for NEF4 function in vivo, and genetic analysis uncovered new interactions between NEF4 and Rad23, a repair factor that links repair to proteasome function. Elc1 is the yeast homologue of a mammalian E3 subunit, and it is a novel component of NEF4. Moreover, the E2s Ubc9 and Ubc13 were linked to the NEF4 repair pathway by genetic criteria. Mutations in NEF4 or Ubc13 result in elevated levels of the DNA damage recognition protein Rad4 and an increase in ubiquitylated species of Rad23. As Rad23 also controls Rad4 levels, these results suggest a complex system for globally regulating repair activity in vivo by controlling turnover of Rad4.

The initiative role of XPC protein in cisplatin DNA damaging treatment-mediated cell cycle regulation.

XPC is an important DNA damage recognition protein involved in DNA nucleotide excision repair. We have studied the role of the XPC protein in cisplatin treatment-mediated cell cycle regulation. Through the comparison of microarray data obtained from human normal fibroblasts and two individual XPC-defective cell lines, 486 genes were identified as XPC-responsive genes in the cisplatin treatment (with a minimal 1.5-fold change) and 297 of these genes were further mapped to biological pathways and gene ontologies. The cell cycle and cell proliferation-related genes were the most affected genes by the XPC defect in the cisplatin treatment. Many other cellular function genes were also affected by the XPC defect in the treatment. Western blot hybridization results revealed that the XPC defect reduced the p53 responses to the cisplatin treatment. The ability to activate caspase-3 was also attenuated in the XPC cells with the treatment. These results suggest that the XPC protein plays a critical role in initiating the cisplatin DNA damaging treatment-mediated signal transduction process, resulting in activation of the p53 pathway and cell cycle arrest that allow DNA repair and apoptosis to take place. These results reveal an important role of the XPC protein in the cancer prevention.

Defining the function of XPC protein in psoralen and cisplatin-mediated DNA repair and mutagenesis.

DNA damage recognition plays an important role in DNA repair and mutagenesis. Failure to recognize DNA damage may lead to DNA replication without damage repair as well as mutation accumulation. Mutations can lead to many disease conditions. XPC is a DNA damage recognition protein that binds to damaged DNA templates at a very early stage during the DNA repair process. We have studied the role of the XPC protein in DNA cross-link reagents, psoralen and cisplatin, mediated DNA repair and mutagenesis. When psoralen and cisplatin-damaged plasmid DNA was transfected into xeroderma pigmentosum group C (XPC) cells, which were defective in the XPC gene, very distinct mutation frequency and spectrum was observed: a decreased mutation frequency for psoralen-damaged plasmid and an increased mutation frequency for cisplatin-damaged plasmid; in contrast, most mutations generated by psoralen in XPC cells were T-to-G transversions and most mutations generated by cisplatin in XPC cells were large deletions. We also determined the DNA repair ability of XPC cells by both host cell reactivation (HCR) assay and in vitro DNA repair assay. The HCR results showed greatly reduced host cell reactivation of a luciferase reporter for both psoralen and cisplatin-damaged plasmid DNA in XPC cells. The in vitro DNA repair results revealed a defective repair capacity for both psoralen and cisplatin-damaged plasmid DNA in nuclear extract prepared from XPC cells. However, this defective DNA repair activity was partially restored when a functional XPC protein was supplemented into the XPC nuclear extract prior to the reaction. These results suggest that the XPC protein DNA damage recognition function plays a crucial role in DNA repair initiation and mutation avoidance and XPC defects may lead to increased mutations and high risk for disease progression.

MutS-DNA interactions and DNase protection analysis with surface plasmon resonance.

Elucidation of the mechanisms of recognition of mispaired DNA by MutS and subsequent repair by the multicomponent mismatch repair machinery presents a fascinating challenge (). ATP binding or hydrolysis are implicated in several steps of the process, including enabling MutS to dissociate from the mismatch, probably through a conformational change () and for MutSL-DNA interactions (,). In addition to mismatch recognition, the protein has significant affinity for normal DNA. This may be important for the protein to scan DNA in search of a mismatch and for its proposed translocation activity. It can be expected, and especially once a MutS-DNA complex structure is solved, that numerous mutants will be generated to study the recognition mechanism. To examine stable and transient DNA interactions of the native and mutant proteins in various conformational states, assays that enable detailed kinetics are desirable. Instrumentation providing such capability is a worthwhile complement to classic assays, such as filter binding and electrophoretic mobility shift assays. Here, we describe techniques for studying the interaction of MutS and MutL with immobilized DNA using surface plasmon resonance and the BIAcore instrument. Such techniques should be readily adaptable to studies of other mismatch binding proteins (MMBP) or other DNA damage recognition proteins.

Specificity of platinum–DNA adduct repair

Cell lines with resistance to cisplatin and carboplatin often retain sensitivity to platinum complexes with different carrier ligands (e.g., oxaliplatin and JM216). HeLa cell extracts were shown to excise cisplatin, oxaliplatin, and JM216 adducts with equal efficiency, suggesting that nucleotide excision repair does not contribute to the carrier-ligand specificity of platinum resistance. We have shown previously that the extent of replicative bypass in vivo is influenced by the carrier ligand of the platinum adducts. The specificity of replicative bypass may be determined by the DNA polymerase complexes that catalyze translesion synthesis past Pt–DNA adducts, by the mismatch-repair system that removes newly synthesized DNA opposite Pt–DNA adducts, and/or by DNA damage-recognition proteins that bind to the Pt–DNA adducts and block translesion synthesis. Primer extension on DNA templates containing site-specifically placed cisplatin, oxaliplatin, or JM216 Pt–GG adducts revealed that the eukaryotic DNA polymerases β , ζ , γ and HIV-1 RT had a similar specificity for translesion synthesis past Pt–DNA adducts (oxaliplatin≥cisplatin>JM216). In addition, defects in the mismatch-repair proteins hMSH6 and hMLH1 led to increased replicative bypass of cisplatin adducts, but not of oxaliplatin adducts. Finally, primer extension assays performed in the presence of HMG1, which is known to recognize cisplatin-damaged DNA, revealed that inhibition of translesion synthesis by HMG1 also depended on the carrier ligand of the Pt–DNA adduct (cisplatin>oxaliplatin=JM216). These studies show that DNA polymerases, the mismatch-repair system and damage-recognition proteins can all impart specificity to replicative bypass of Pt–DNA adducts. Replicative bypass, in turn, may influence the carrier-ligand specificity of resistance.

Poly(ADP-ribosyl)ation is required for p53-dependent signal transduction induced by radiation.

p53 and poly(ADP-ribose) polymerase (PARP) are both DNA damage recognition proteins and can be functionally activated by DNA strand breaks. To understand the functional interaction between these two proteins, the effects of a PARP inhibitor, 3-aminobenzamide (3AB), on the p53 pathway were investigated in human glioblastoma cells with different p53 status. Consistent with previous studies, irradiation with gamma-rays induced both p53 and WAF1 accumulation in A-172 cells (wtp53) but not in T98G cells (mp53). However, the presence of 3AB but not its analog suppressed radiation-induced accumulation of wtp53 and the expression of WAF1 and MDM2. Similar results were also obtained from U87MG, another human glioblastoma cell line with wtp53 status. Northern blotting analysis showed that 3AB inhibited the gamma-ray-induced WAF1 gene expression. Moreover, 3AB but not its analog inhibited irradiation-induced activation of sequence-specific DNA binding of wtp53 as detected using 32P-labeled or biotin-labeled p53 consensus sequence (p53CON). However, immunoblotting with an anti-poly(ADP-ribose) antibody showed that p53 proteins of the p53CON-bound fraction did not contain poly(ADP-ribose) (PAR). These findings suggested that poly(ADP-ribosyl)ation is required for rapid accumulation of p53, activation of p53 sequence-specific DNA binding and its transcriptional activity after DNA damage.

Affinity Purification and Partial Characterization of a Yeast Multiprotein Complex for Nucleotide Excision Repair Using Histidine-tagged Rad14 Protein

The nucleotide excision repair (NER) pathway of eukaryotes involves approximately 30 polypeptides. Reconstitution of this pathway with purified components is consistent with the sequential assembly of NER proteins at the DNA lesion. However, recent studies have suggested that NER proteins may be pre-assembled in a high molecular weight complex in the absence of DNA damage. To examine this model further, we have constructed a histidine-tagged version of the yeast DNA damage recognition protein Rad14. Affinity purification of this protein from yeast nuclear extracts resulted in the co-purification of Rad1, Rad7, Rad10, Rad16, Rad23, RPA, RPB1, and TFIIH proteins, whereas none of these proteins bound to the affinity resin in the absence of recombinant Rad14. Furthermore, many of the co-purifying proteins were present in approximately equimolar amounts. Co-elution of these proteins was also observed when the nuclear extract was fractionated by gel filtration, indicating that the NER proteins were associated in a complex with a molecular mass of >1000 kDa prior to affinity chromatography. The affinity purified NER complex catalyzed the incision of UV-irradiated DNA in an ATP-dependent reaction. We conclude that active high molecular weight complexes of NER proteins exist in undamaged yeast cells.