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.     

Pre-expression of a sulfhydryl oxidase significantly increases the yields of eukaryotic disulfide bond containing proteins expressed in the cytoplasm of E.coli

Background  

Disulfide bonds are one of the most common post-translational modifications found in proteins. The production of proteins that contain native disulfide bonds is challenging, especially on a large scale. Either the protein needs to be targeted to the endoplasmic reticulum in eukaryotes or to the prokaryotic periplasm. These compartments that are specialised for disulfide bond formation have an active catalyst for their formation, along with catalysts for isomerization to the native state. We have recently shown that it is possible to produce large amounts of prokaryotic disulfide bond containing proteins in the cytoplasm of wild-type bacteria such as E. coli by the introduction of catalysts for both of these processes.     

4-Thiothymidine sensitization of DNA to UVA offers potential for a novel photochemotherapy

Photochemotherapy, in which ultraviolet radiation (UVR: 280-400 nm) or visible light is combined with a photosensitizing drug to produce a therapeutic effect that neither drug or radiation can achieve alone, is a proven therapeutic strategy for a number of non-malignant hyperproliferative skin conditions and various cancers. Examples are psoralen plus UVA (320-400 nm) radiation (PUVA) and photodynamic therapy (PDT). All existing photochemotherapies have drawbacks - for example the association of PUVA with the development of skin cancer, and pain that is often associated with PDT treatment of skin lesions. There is a clear need to develop alternative approaches that involve lower radiation doses and/or improved selectivity for target cells. In this review, we explore the possibility to address this need by exploiting thionucleoside-mediated DNA photosensitisation to low, non toxic doses of UVA radiation.     

Ambiguous coding is required for the lethal interaction between methylated DNA bases and DNA mismatch repair

The thiopurine 6-thioguanine (S6G) is used to treat acute leukaemia. Its cytotoxic effect requires an active DNA mismatch repair (MMR) system. S6G is incorporated into DNA where a small fraction undergoes in situ conversion to S6-thiomethylguanine (S6meG). After replication, S6meG-containing base pairs interact with MMR. This interaction is ultimately lethal and MMR-defective cells are resistant to S6G. Here, we report that growing human cells extensively incorporate the thiopyrimidine nucleoside 4-thiothymidine (S4TdR) into their DNA. The incorporated thiopyrimidine (S4T) can also undergo facile S-methylation to 4-thiomethylthymine (S4meT). The rate of methylation of S4TdR in model substrates is similar to that for the conversion of S6G to S6meG indicating that the DNA of cells grown in S4TdR will contain significant levels of S4meT. Despite this, S4TdR is not associated with MMR-related cell death. We demonstrate that, in contrast to S6meG, neither DNA S4T nor S4meT codes ambiguously. S4T retains the coding properties of unmodified T, whereas S4meT behaves like a normal cytosine and exclusively directs the incorporation of guanine. The preferred S4meT:G base pair is also a poor substrate for binding by the hMutSalpha mismatch recognition factor. We suggest that the ability of S4meT to produce a structurally acceptable base pair during replication underlies the absence of MMR-related death in cells treated with S4TdR.     

A model of dengue fever

Background  

Dengue is a disease which is now endemic in more than 100 countries of Africa, America, Asia and the Western Pacific. It is transmitted to the man by mosquitoes (Aedes) and exists in two forms: Dengue Fever and Dengue Haemorrhagic Fever. The disease can be contracted by one of the four different viruses. Moreover, immunity is acquired only to the serotype contracted and a contact with a second serotype becomes more dangerous.     

Structural and dynamic effects of single 7-hydro-8-oxoguanine bases located in a frameshift target DNA sequence

DNA 7-hydro-8-oxoguanine (8-oxoG) is implicated in frameshift formation in an G(6) sequence of the HPRT gene in mismatch repair (MMR) defective cells. Using oligonucleotides based on this frameshift hotspot, we investigated how a single 8-oxoG modified the structural and dynamic properties of the G(6) tract. A 30 ns molecular dynamics (MD) simulation indicated compression of the minor groove in the immediate vicinity of the lesion. Fluorescence polarization anisotropy (FPA) and MD demonstrated that 8-oxoG increases DNA torsional rigidity and also constrains the movement of the single-stranded region at the single/double stranded DNA junction of model DNA replication template/primer. These constraints influenced the efficiency of primer extension by Klenow (exo(-)) DNA polymerase.     

Selective binding to DNA base pair mismatches by proteins from human cells

Using the technique of delayed oligonucleotide migration through polyacrylamide gels, we have demonstrated that cell-free extracts of the human Burkitt's lymphoma cell line Raji contain proteins which can recognize and bind to mismatched single base pairs in short fragments of DNA. One of these binding proteins resembles an activity previously reported in HeLa cells (Jiricny, J., Hughes, M., Corman, N., and Rudkin, B. B. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8860-8864) and recognizes DNA containing G.T mismatches. Extracts of Raji cells contain an additional activity which recognizes A.C, T.C, or T.T mismatches in DNA. This second binding protein can be distinguished from the G.T binding activity by its size, substrate specificity, and its fractionation properties. In addition to Raji cells, the new mismatch binding protein is present in extracts of human lymphoblastoid cell lines from a normal individual and a xeroderma pigmentosum patient as well as the SV40-transformed human fibroblast cell line MRC5V1. It seems likely that this novel activity is involved in a broad specificity DNA repair pathway for the correction of single base mismatches in human cells.     

Inactive O6-methylguanine-DNA methyltransferase in human cells.

A plasmid encoding a recombinant human O6-methylguanine-DNA methyltransferase (MGMT) fused to a fragment of the bacteriophage lambda N protein has been constructed. The fusion protein retained methyltransferase activity when expressed at high levels in E.coli and was purified to essential homogeneity by a simple procedure. Antisera raised against the purified fusion protein recognized MGMT in western blots of extracts of human cells. For most cell lines, there was a quantitative relation between the amount of immunologically detectable MGMT protein and enzyme activity. However, four cell lines contained detectable MGMT protein despite having no measurable methyltransferase activity. Additionally, a HeLa line contained considerably more immunoreactive MGMT protein than could be accounted for by its methyltransferase activity. Thus, some cells contain significant amounts of inactive MGMT. Preliminary characterization of the inactive protein in HeLaS3 cells indicated that it has some properties in common with MGMT methylated at the active cysteine residue.