Photo-cross-linking of XPC-Rad23B to cisplatin-damaged DNA reveals contacts with both strands of the DNA duplex and spans the DNA adduct.

Nucleotide excision repair (NER) is the main pathway used for the repair of bulky DNA adducts such as those caused by UV light exposure and the chemotherapeutic drug cisplatin. The xeroderma pigmentosum group C (XPC)-Rad23B complex is involved in the recognition of these bulky DNA adducts and initiates the global genomic nucleotide excision repair pathway (GG-NER). Photo-cross-linking experiments revealed that the human XPC-Rad23B complex makes direct contact with both the cisplatin-damaged DNA strand and the complementary undamaged strand of a duplex DNA substrate. Coupling photo-cross-linking with denaturation and immunoprecipitation of protein-DNA complexes, we identified the XPC subunit in complex with damaged DNA. While the interaction of the XPC subunit with DNA was direct, studies revealed that although Rad23B was found in complex with DNA, the Rad23B-DNA interaction was largely indirect via its interaction with XPC. Using site specific cross-linking, we determined that the XPC-Rad23B complex is preferentially cross-linked to the damaged DNA when the photoreactive FAP-dCMP (exo-N-{2-[N-(4-azido-2,5-difluoro-3-chloropyridin-6-yl)-3-aminopropionyl]aminoethyl}-2'-deoxycytidine 5'-monophosphate) analogue is located to the 5' side of the cisplatin-DNA adduct. When the FAP-dCMP analogue is located to the 3' side of the adduct, no difference in binding was detected between undamaged and damaged DNA. Collectively, these data suggest a model in which XPC-DNA interactions drive the damage recognition process contacting both the damaged and undamaged DNA strand. Preferential cross-linking 5' of the cisplatin-damaged site suggests that the XPC-Rad23B complex displays orientation specific binding to eventually impart directionality to the downstream binding and incision events relative to the site of DNA damage.

Molecular analysis of Ku redox regulation.

BACKGROUND: DNA double-strand breaks (DSBs) can occur in response to ionizing radiation (IR), radiomimetic agents and from endogenous DNA-damaging reactive oxygen metabolites. Unrepaired or improperly repaired DSBs are potentially the most lethal form of DNA damage and can result in chromosomal translocations and contribute to the development of cancer. The principal mechanism for the repair of DSBs in humans is non-homologous end-joining (NHEJ). Ku is a key member of the NHEJ pathway and plays an important role in the recognition step when it binds to free DNA termini. Ku then stimulates the assembly and activation of other NHEJ components. DNA binding of Ku is regulated by redox conditions and evidence from our laboratory has demonstrated that Ku undergoes structural changes when oxidized that results in a reduction in DNA binding activity. The C-terminal domain and cysteine 493 of Ku80 were investigated for their contribution to redox regulation of Ku. RESULTS: We effectively removed the C-terminal domain of Ku80 generating a truncation mutant and co-expressed this variant with wild type Ku70 in an insect cell system to create a Ku70/80DeltaC heterodimer. We also generated two single amino acid variants of Cys493, replacing this amino acid with either an alanine (C493A) or a serine (C493S), and over-expressed the variant proteins in SF9 insect cells in complex with wild type Ku70. Neither the truncation nor the amino acid substitutions alters protein expression or stability as determined by SDS-PAGE and Western blot analysis. We show that the C493 mutations do not alter the ability of Ku to bind duplex DNA in vitro under reduced conditions while truncation of the Ku80 C-terminus slightly reduced DNA binding affinity. Diamide oxidation of cysteines was shown to inhibit DNA binding similarly for both the wild-type and all variant proteins. Interestingly, differential DNA binding activity following re-reduction was observed for the Ku70/80DeltaC truncation mutant. CONCLUSION: Together, these results suggest that the C-terminal domain and C493 of Ku80 play at most a minor role in the redox regulation of Ku, and that other cysteines are likely involved, either alone or in conjunction with these regions of Ku80.

Pseudomonas fluorescens ompW: plasmid localization and requirement for naphthalene uptake.

Suppressive subtractive hybridization has been utilized to generate a cDNA library of genes differentially expressed in naphthalene grown cells of Pseudomonas fluorescens. The library was devoid of genes known to be associated with naphthalene catabolism, but was enriched in genes related to cellular uptake and efflux systems. The gene for OmpW, which was present in the cDNA library and has been proposed to encode a porin for the transport of hydrophobic molecules, was isolated, cloned, and sequenced. This gene was shown to be exclusively localized on a large catabolic plasmid possessed by the organism, and its specific mutation resulted in the loss of the organism's ability to grow on naphthalene and several other polycyclic aromatic hydrocarbons. It is proposed that a primary response by P. fluorescens to the presence of naphthalene is the elevation of the cellular mechanism(s) required for its assimilation. The presence of genes related to the uptake and efflux mechanisms present following suppressive subtractive hybridization supports this proposal.

Current advances in DNA repair: regulation of enzymes and pathways involved in maintaining genomic stability.

Novel discoveries in the DNA repair field have lead to continuous and rapid advancement of our understanding of not only DNA repair but also DNA replication and recombination. Research in the field transcends numerous areas of biology, biochemistry, physiology, and medicine, making significant connections across these broad areas of study. From early studies conducted in bacterial systems to current analyses in eukaryotic systems and human disease, the innovative research into the mechanisms of repair machines and the consequences of ineffective DNA repair has impacted a wide scientific community. This Forum contains a select mix of primary research articles in addition to a number of timely reviews covering a subset of DNA repair pathways where recent advances and novel discoveries are improving our understanding of DNA repair, its regulation, and implications to human disease.

Novel irreversible small molecule inhibitors of replication protein A display single-agent activity and synergize with cisplatin.

Replication protein A (RPA) is a single-strand DNA-binding protein with essential roles in DNA replication, recombination, and repair. It is necessary for the formation of the preincision complex that is required for proper incision of damaged DNA nucleotides during DNA repair. We have previously identified small molecule inhibitors (SMI) with the ability to disrupt RPA-binding activity to ssDNA. Further characterization of these RPA inhibitors was done using both lung and ovarian cancer cell lines. Lung cancer cell lines showed increased apoptotic cell death following treatment with the SMI MCI13E, with IC(50) values of approximately 5 µmol/L. The ovarian cancer cell line A2780 and the p53-null lung cancer cell line H1299 were particularly sensitive to MCI13E treatment, with IC(50) values less than 3 µmol/L. Furthermore, a cell-cycle effect was observed in lung cancer cell lines that resulted in a lengthening of either G(1) or S-phases of the cell cycle following single-agent treatment. Sequential treatment with MCI13E and cisplatin resulted in synergism. Overall, these data suggest that decreasing DNA-binding activity of RPA via a SMI may disrupt the role of RPA in cell-cycle regulation. Thus, SMIs of RPA hold the potential to be used as single-agent chemotherapeutics or in combination with current chemotherapeutic regimens to increase efficacy.

Identification of novel small molecule inhibitors of the XPA protein using in silico based screening.

The nucleotide excision repair pathway catalyzes the removal of bulky adduct damage from DNA and requires the activity of more than 30 individual proteins and complexes. A diverse array of damage can be recognized and removed by the NER pathway including UV-induced adducts and intrastrand adducts induced by the chemotherapeutic compound cisplatin. The recognition of DNA damage is complex and involves a series of proteins including the xeroderma pigmentosum group A and C proteins and the UV-damage DNA binding protein. The xeroderma pigmentosum group A protein is unique in the sense that it is required for both transcription coupled and global genomic nucleotide excision repair. In addition, xeroderma pigmentosum group A protein is required for the removal of all types of DNA lesions repaired by nucleotide excision repair. Considering its importance in the damage recognition process, the minimal information available on the mechanism of DNA binding, and the potential that inhibition of xeroderma pigmentosum group A protein could enhance the therapeutic efficacy of platinum based anticancer drugs, we sought to identify and characterize small molecule inhibitors of the DNA binding activity of the xeroderma pigmentosum group A protein. In silico screening of a virtual small molecule library resulted in the identification of a class of molecules confirmed to inhibit the xeroderma pigmentosum group A protein-DNA interaction. Biochemical analysis of inhibition with varying DNA substrates revealed a common mechanism of xeroderma pigmentosum group A protein DNA binding to single-stranded DNA and cisplatin-damaged DNA.