Toshiro Matsuda

Low fidelity DNA synthesis by human DNA polymerase-eta.

A superfamily of DNA polymerases that bypass lesions in DNA has been described. Some family members are described as error-prone because mutations that inactivate the polymerase reduce damage-induced mutagenesis. In contrast, mutations in the skin cancer susceptibility gene XPV, which encodes DNA polymerase (pol)-η, lead to increased ultraviolet-induced mutagenesis. This, and the fact that pol-η primarily inserts adenines during efficient bypass of thymine–thymine dimers in vitro, has led to the description of pol-η as error-free. However, here we show that human pol-η copies undamaged DNA with much lower fidelity than any other template-dependent DNA polymerase studied. Pol-η lacks an intrinsic proofreading exonuclease activity and, depending on the mismatch, makes one base substitution error for every 18 to 380 nucleotides synthesized. This very low fidelity indicates a relaxed requirement for correct base pairing geometry and indicates that the function of pol-η may be tightly controlled to prevent potentially mutagenic DNA synthesis.

Somatic mutation hotspots correlate with DNA polymerase |[eta]| error spectrum

Mutational spectra analysis of 15 immunoglobulin genes suggested that consensus motifs RGYW and WA were universal descriptors of somatic hypermutation. Highly mutable sites, “hotspots”, that matched WA were preferentially found in one DNA strand and RGYW hotspots were found in both strands. Analysis of base-substitution hotspots in DNA polymerase error spectra showed that 33 of 36 hotspots in the human polymerase η spectrum conformed to the WA consensus. This and four other characteristics of polymerase η substitution specificity suggest that errors introduced by this enzyme during synthesis of the nontranscribed DNA strand in variable regions may contribute to strand-specific somatic hypermutagenesis of immunoglobulin genes at A-T base pairs.

XAB2, a Novel Tetratricopeptide Repeat Protein Involved in Transcription-coupled DNA Repair and Transcription

Nucleotide excision repair is a highly versatile DNA repair system responsible for elimination of a wide variety of lesions from the genome. It is comprised of two subpathways: transcription-coupled repair that accomplishes efficient removal of damage blocking transcription and global genome repair. Recently, the basic mechanism of global genome repair has emerged from biochemical studies. However, little is known about transcription-coupled repair in eukaryotes. Here we report the identification of a novel protein designated XAB2 (XPA-binding protein 2) that was identified by virtue of its ability to interact with XPA, a factor central to both nucleotide excision repair subpathways. The XAB2 protein of 855 amino acids consists mainly of 15 tetratricopeptide repeats. In addition to interacting with XPA, immunoprecipitation experiments demonstrated that a fraction of XAB2 is able to interact with the transcription-coupled repair-specific proteins CSA and CSB as well as RNA polymerase II. Furthermore, antibodies against XAB2 inhibited both transcription-coupled repair and transcription in vivo but not global genome repair when microinjected into living fibroblasts. These results indicate that XAB2 is a novel component involved in transcription-coupled repair and transcription.

Identification of a damaged-DNA binding domain of the XPA protein

The XPA (xeroderma pigmentosum group A) protein is a zinc metalloprotein consisting of 273 amino acids which binds preferentially to UV- or chemical carcinogen-damaged DNA, suggesting that it is involved in the recognition of several types of DNA damage during nucleotide excision repair processes. Here we identify a DNA binding domain of the XPA protein. The region of the XPA protein responsible for preferential binding to DNA damaged by UV or cis-diammine-dichloroplatinum(II) (cisplatin) is contained within a truncated derivative of the XPA protein, MF122, consisting of 122 amino acids and containing a C4 type zinc finger motif. CD (circular dichroism) measurements of the MF122 protein showed that it has a helix-rich secondary structure, suggesting that it is a discretely folded, functional mini-domain. The MF122 protein should be useful for structural investigation of the XPA protein and of its interaction with damaged DNA.

Geometric dependence of the energy confinement time scaling for H-mode discharges

The dependence of the H-mode energy confinement time τE on the plasma elongation and the divertor configuration is studied. A regression analysis of the H-mode database is presented in which the dependence on the closed/open divertor configuration, estimated from the ASDEX experiments, is included. The dependence of τE on the elongation factor k is found to be positive (approximately k0.6) for ELM-free H-mode discharges, in accordance with experimental results for ELM-free limiter H-mode plasmas in JFT-2M. In addition, the influence of the X-point configuration (single null or double null) on the confinement time is briefly addressed

Mitotic genes are transcriptionally upregulated in the fibroblast irradiated with very low doses of UV-C

Ultraviolet (UV) radiation induces a variety of biological effects, including DNA damage response and cell signaling pathways. We performed transcriptome analysis using microarray in human primary cultured fibroblasts irradiated with UV-C (0.5 or 5 J/m2) and harvested at 4 or 12 h following UV exposure. All transcript data were analyzed by comparison with the corresponding results in non-irradiated (control) cells. The number of genes with significantly altered expression (≥2-fold difference relative to the control) is higher in the sample irradiated with high dose of UV, suggesting that gene expression was UV dose-dependent. Pathway analysis on the upregulated genes at 12 h indicates that the expression of some cell cycle-related genes was predominantly induced irrespective of UV-dose. Interestingly, almost all the genes with significant altered expression were cell cycle-related genes designated as ‘Mitotic Genes’, which function in the spindle assembly checkpoint. Therefore, even a low dose of UV could affect the transcriptional profile.