Protein oxidation and DNA repair inhibition by 6-thioguanine and UVA radiation.

Damage to skin DNA by solar UV is largely unavoidable, and an optimal cellular response to it requires the coordinated operation of proteins in numerous pathways. A fully functional DNA repair proteome for removing harmful DNA lesions is a prerequisite for an appropriate DNA damage response. Genetically determined failure to repair UV-induced DNA damage is associated with skin photosensitivity and increased skin cancer risk. Patients treated with immunosuppressant/anti-inflammatory thiopurines are also photosensitive and have high rates of sun-related skin cancer. Their DNA contains the base analog 6-thioguanine (6-TG), which acts as a UVA photosensitizer to generate reactive oxygen species (ROS), predominantly singlet oxygen ((1)O2). ROS damage both DNA and proteins. Here we show that UVA irradiation of cultured human cells containing DNA 6-TG causes significant protein oxidation and damages components of the DNA repair proteome, including the Ku, OGG-1, MYH, and RPA proteins. Assays of DNA repair in intact cells or in cell extracts indicate that this protein damage compromises DNA break rejoining and base and nucleotide excision repair. As these experimental conditions simulate those in the skin of patients taking thiopurines, our findings suggest a mechanism whereby UVA in sunlight may contribute to skin carcinogenesis in immunosuppressed patients.

Crosslinking of DNA repair and replication proteins to DNA in cells treated with 6-thioguanine and UVA.

The DNA of patients taking immunosuppressive and anti-inflammatory thiopurines contains 6-thioguanine (6-TG) and their skin is hypersensitive to ultraviolet A (UVA) radiation. DNA 6-TG absorbs UVA and generates reactive oxygen species that damage DNA and proteins. Here, we show that the DNA damage includes covalent DNA-protein crosslinks. An oligonucleotide containing a single 6-TG is photochemically crosslinked to cysteine-containing oligopeptides by low doses of UVA. Crosslinking is significantly more efficient if guanine sulphonate (G(SO3))--an oxidized 6-TG and a previously identified UVA photoproduct--replaces 6-TG, suggesting that G(SO3) is an important reaction intermediate. Crosslinking occurs via oligopeptide sulphydryl and free amino groups. The oligonucleotide-oligopeptide adducts are heat stable but are partially reversed by reducing treatments. UVA irradiation of human cells containing DNA 6-TG induces extensive heat- and reducing agent-resistant covalent DNA-protein crosslinks and diminishes the recovery of some DNA repair and replication proteins from nuclear extracts. DNA-protein crosslinked material has an altered buoyant density and can be purified by banding in cesium chloride (CsCl) gradients. PCNA, the MSH2 mismatch repair protein and the XPA nucleotide excision repair (NER) factor are among the proteins detectable in the DNA-crosslinked material. These findings suggest that the 6-TG/UVA combination might compromise DNA repair by sequestering essential proteins.

Differential roles for DNA polymerases eta, zeta, and REV1 in lesion bypass of intrastrand versus interstrand DNA cross-links.

Translesion DNA synthesis (TLS) is a process whereby specialized DNA polymerases are recruited to bypass DNA lesions that would otherwise stall high-fidelity polymerases. We provide evidence that TLS across cisplatin intrastrand cross-links is performed by multiple translesion DNA polymerases. First, we determined that PCNA monoubiquitination by RAD18 is necessary for efficient bypass of cisplatin adducts by the TLS polymerases eta (Poleta), REV1, and zeta (Polzeta) based on the observations that depletion of these proteins individually leads to decreased cell survival, cell cycle arrest in S phase, and activation of the DNA damage response. Second, we showed that in addition to PCNA monoubiquitination by RAD18, the Fanconi anemia core complex is also important for recruitment of REV1 to stalled replication forks in cisplatin treated cells. Third, we present evidence that REV1 and Polzeta are uniquely associated with protection against cisplatin and mitomycin C-induced chromosomal aberrations, and both are necessary for the timely resolution of DNA double-strand breaks associated with repair of DNA interstrand cross-links. Together, our findings indicate that REV1 and Polzeta facilitate repair of interstrand cross-links independently of PCNA monoubiquitination and Poleta, whereas RAD18 plus Poleta, REV1, and Polzeta are all necessary for replicative bypass of cisplatin intrastrand DNA cross-links.

Competitive repair pathways in immunoglobulin gene hypermutation.

This review focuses on the contribution of translesion DNA polymerases to immunoglobulin gene hypermutation, in particular on the roles of DNA polymerase eta (Poleta) in the generation of mutations at A/T bases from the initial cytosine-targeted activation-induced cytidine deaminase (AID)-mediated deamination event, and of Polkappa, an enzyme of the same polymerase family, used as a substitute when Poleta is absent. The proposition that the UNG uracil glycosylase and the MSH2-MSH6 mismatch recognition complex are two competitive rather than alternative pathways in the processing of uracils generated by AID is further discussed.

Reduced efficiency and increased mutagenicity of translesion DNA synthesis across a TT cyclobutane pyrimidine dimer, but not a TT 6-4 photoproduct, in human cells lacking DNA polymerase eta.

Xeroderma pigmentosum variant (XPV) patients carry germ-line mutations in DNA polymerase eta (poleta), a major translesion DNA synthesis (TLS) polymerase, and exhibit severe sunlight sensitivity and high predisposition to skin cancer. Using a quantitative TLS assay system based on gapped plasmids we analyzed TLS across a site-specific TT CPD (thymine-thymine cyclobutane pyrimidine dimer) or TT 6-4 PP (thymine-thymine 6-4 photoproduct) in three pairs of poleta-proficient and deficient human cells. TLS across the TT CPD lesion was reduced by 2.6-4.4-fold in cells lacking poleta, and exhibited a strong 6-17-fold increase in mutation frequency at the TT CPD. All targeted mutations (74%) in poleta-deficient cells were opposite the 3'T of the CPD, however, a significant fraction (23%) were semi-targeted to the nearest nucleotides flanking the CPD. Deletions and insertions were observed at a low frequency, which increased in the absence of poleta, consistent with the formation of double strand breaks due to defective TLS. TLS across TT 6-4 PP was about twofold lower than across CPD, and was marginally reduced in poleta-deficient cells. TLS across TT 6-4 PP was highly mutagenic (27-63%), with multiple mutations types, and no significant difference between cells with or without poleta. Approximately 50% of the mutations formed were semi-targeted, of which 84-93% were due to the insertion of an A opposite the template G 5' to the 6-4 PP. These results, which are consistent with the UV hyper-mutability of XPV cells, highlight the critical role of poleta in error-free TLS across CPD in human cells, and suggest a potential involvement, although minor, of poleta in TLS across 6-4 PP under some conditions.

Role of DNA polymerases eta, iota and zeta in UV resistance and UV-induced mutagenesis in a human cell line.

Genes coding for DNA polymerases eta, iota and zeta, or for both Pol eta and Pol iota have been inactivated by homologous recombination in the Burkitt's lymphoma BL2 cell line, thus providing for the first time the total suppression of these enzymes in a human context. The UV sensitivities and UV-induced mutagenesis on an irradiated shuttle vector have been analyzed for these deficient cell lines. The double Pol eta/iota deficient cell line was more UV sensitive than the Pol eta-deficient cell line and mutation hotspots specific to the Pol eta-deficient context appeared to require the presence of Pol iota, thus strengthening the view that Pol iota is involved in UV damage translesion synthesis and UV-induced mutagenesis. A role for Pol zeta in a damage repair process at late replicative stages is reported, which may explain the drastic UV-sensitivity phenotype observed when this polymerase is absent. A specific mutation pattern was observed for the UV-irradiated shuttle vector transfected in Pol zeta-deficient cell lines, which, in contrast to mutagenesis at the HPRT locus previously reported, strikingly resembled mutations observed in UV-induced skin cancers in humans. Finally, a Pol eta PIP-box mutant (without its PCNA binding domain) could completely restore the UV resistance in a Pol eta deficient cell line, in the absence of UV-induced foci, suggesting, as observed for Pol iota in a Pol eta-deficient background, that TLS may occur without the accumulation of microscopically visible repair factories.

Induction of somatic hypermutation in immunoglobulin genes is dependent on DNA polymerase iota.

Somatic hypermutation of immunoglobulin genes is a unique, targeted, adaptive process. While B cells are engaged in germinal centres in T-dependent responses, single base substitutions are introduced in the expressed Vh/Vl genes to allow the selection of mutants with a higher affinity for the immunizing antigen. Almost every possible DNA transaction has been proposed to explain this process, but each of these models includes an error-prone DNA synthesis step that introduces the mutations. The Y family of DNA polymerases--pol eta, pol iota, pol kappa and rev1--are specialized for copying DNA lesions and have high rates of error when copying a normal DNA template. By performing gene inactivation in a Burkitt's lymphoma cell line inducible for hypermutation, we show here that somatic hypermutation is dependent on DNA polymerase iota.

AID-dependent somatic hypermutation occurs as a DNA single-strand event in the BL2 cell line.

Immunoglobulin (Ig) gene hypermutation can be induced in the BL2 Burkitt's lymphoma cell line by IgM cross-linking and coculture with normal or transformed T helper clones. We describe here a T cell#150;independent in vitro induction assay, by which hypermutation is induced in BL2 cells through simultaneous aggregation of three surface receptors: IgM, CD19 and CD21. The mutations arise as a post-transcriptional event within 90 min. They are stably introduced in the G1 phase of the cell cycle, occurring in one of the two variable gene DNA strands, and eventually become fixed by replication in one of the daughter cells. Inactivation of AID (activation-induced cytidine deaminase) by homologous recombination in BL2 cells completely inhibits the process, thus validating this induction procedure as a model for the in vivo mechanism.