Fumio Hanaoka

MSH2–MSH6 stimulates DNA polymerase η, suggesting a role for A:T mutations in antibody genes

Activation-induced cytidine deaminase deaminates cytosine to uracil (dU) in DNA, which leads to mutations at C:G basepairs in immunoglobulin genes during somatic hypermutation. The mechanism that generates mutations at A:T basepairs, however, remains unclear. It appears to require the MSH2–MSH6 mismatch repair heterodimer and DNA polymerase (pol) η, as mutations of A:T are decreased in mice and humans lacking these proteins. Here, we demonstrate that these proteins interact physically and functionally. First, we show that MSH2–MSH6 binds to a U:G mismatch but not to other DNA intermediates produced during base excision repair of dUs, including an abasic site and a deoxyribose phosphate group. Second, MSH2 binds to pol η in solution, and endogenous MSH2 associates with the pol in cell extracts. Third, MSH2–MSH6 stimulates the catalytic activity of pol η in vitro. These observations suggest that the interaction between MSH2–MSH6 and DNA pol η stimulates synthesis of mutations at bases located downstream of the initial dU lesion, including A:T pairs.

Blasticidin S-resistance gene (bsr): a novel selectable marker for mammalian cells.

Blasticidin S is a microbial antibiotic that inhibits protein synthesis in both prokaryotes and eukaryotes. The blasticidin S-resistance gene (bsr), isolated from Bacillus cereus K55-S1 strain, was inserted into pSV2 plasmid vector and introduced into cultured mammalian cells by transfection. The bsr gene was integrated into the genome and conferred blasticidin S resistance on HeLa cells. The transfection frequency of the bsr gene was as high as that of the aminoglycoside phosphotransferase gene, the so-called neo gene, which is a representative selectable marker for mammalian cells. Transfectants in which several copies of bsr had been integrated into the genome were highly resistant to blasticidin S. Furthermore, blasticidin S killed the cells more rapidly than G418, which is conventionally used as a selective drug for the neo gene. Thus bsr is concluded to be useful as a drug-resistance marker for mammalian cells.

A human homologue of the yeast GST1 gene codes for a GTP-binding protein and is expressed in a proliferation-dependent manner in mammalian cells.

A human homologue (GST1‐Hs) of the yeast GST1 gene that encodes a new GTP‐binding protein essential for the G1‐to‐S phase transition of the cell cycle was cloned from the cDNA library of human KB cells. The GST1‐Hs cDNA contained a 1497 bp open reading frame coding for a 499 amino acid protein with mol. wt 55,754 and with the amino acid sequence homologies of 52.3 and 37.8% to the GST1 protein and polypeptide chain elongation factor EF1 alpha respectively. The regions potentially responsible for GTP binding and GTP hydrolysis were conserved in the GST1‐Hs protein as well. When expressed in yeast cell, the GST1‐Hs gene could complement the ts phenotype of yeast gst1 mutant. GST1‐Hs and its mouse homologue were expressed in human fibroblasts and in various mouse cell types respectively, at relatively low levels in their quiescent states, and the level of those expressions increased rapidly, prior to the onset of DNA replication and the total RNA synthesis, when human or mouse fibroblasts were progressed out of the growth‐arrested state by the addition of serum. A possible role of GST1‐Hs in mammalian cell growth is discussed.

Aphidicolin does inhibit repair replication in HeLa cells.

Abstract Aphidicolin was shown to be a specific inhibitor of eukaryotic DNA polymerase α. We have examined the effect of aphidicolin on repair synthesis as well as replication of HeLa cell DNA, and found that it inhibits not only DNA replication but also UV-induced DNA repair in hydroxyurea-arabinosyl cytosine treated cells.

Localization of the replication point of mammalian cell DNA at the membrane

Abstract The replication site of DNA in HeLa S3 cells has been studied with “M-band” technique after Tremblay and others. When the cells are mixed with sodium lauroyl sarcosinate and Magnesium ion directly on the sucrose gradient and spun, virtually all the DNA is caught in the M-band, suggesting that mammalian cell DNA is attached to the nuclear membrane. After being treated with sodium lauroyl sarcosinate and sheared by a vortex-mixer, the DNA is found to have moved from the M-band to the supernatant. Pulse-labeled DNA is more resistant to shearing as compared with the bulk of DNA and chased away from the M-band during a subsequent growth. These results indicate that, as in the case of bacteria, the replication points of mammalian cell DNA are on the membrane.

Structural basis of human DNA polymerase η-mediated chemoresistance to cisplatin

Cisplatin (cis-diamminedichloroplatinum) and related compounds cause DNA damage and are widely used as anticancer agents. Chemoresistance to cisplatin treatment is due in part to translesion synthesis by human DNA polymerase η (hPol η). Here, we report crystal structures of hPol η complexed with intrastrand cisplatin-1,2–cross-linked DNA, representing four consecutive steps in translesion synthesis. In contrast to the generally enlarged and nondiscriminating active site of Y-family polymerases like Dpo4, Pol η is specialized for efficient bypass of UV–cross-linked pyrimidine dimers. Human Pol η differs from the yeast homolog in its binding of DNA template. To incorporate deoxycytidine opposite cisplatin–cross-linked guanines, hPol η undergoes a specific backbone rearrangement to accommodate the larger base dimer and minimizes the DNA distortion around the lesion. Our structural analyses show why Pol η is inefficient at extending primers after cisplatin lesions, which necessitates a second translesion DNA polymerase to complete bypass in vivo. A hydrophobic pocket near the primer-binding site in human Pol η is identified as a potential drug target for inhibiting translesion synthesis and, thereby, reducing chemoresistance.

Complementation of defective translesion synthesis and UV light sensitivity in xeroderma pigmentosum variant cells by human and mouse DNA polymerase η

Defects in the human gene XPV result in the variant form of the genetic disease xeroderma pigmentosum (XP-V). XPV encodes DNA polymerase eta, a novel DNA polymerase that belongs to the UmuC/DinB/Rad30 superfamily. This polymerase catalyzes the efficient and accurate translesion synthesis of DNA past cis-syn cyclobutane di-thymine lesions. In this report we present the cDNA sequence and expression profiles of the mouse XPV gene and demonstrate its ability to complement defective DNA synthesis in XP-V cells. The mouse XPV protein shares 80.3% amino acid identity and 86.9% similarity with the human XPV protein. The recombinant mouse XPV protein corrected the inability of XP-V cell extracts to carry out DNA replication, by bypassing thymine dimers on template DNA. Transfection of the mouse or human XPV cDNA into human XP-V cells corrected UV sensitivity. Northern blot analysis revealed that the mouse XPV gene is expressed ubiquitously, but at a higher level in testis, liver, skin and thymus compared to other tissues. Although the mouse XPV gene was not induced by UV irradiation, its expression was elevated approximately 4-fold during cell proliferation. These results suggest that DNA polymerase eta plays a role in DNA replication, though the enzyme is not essential for viability.

Altered frequency of initiation sites of DNA replication in Werner's syndrome cells

SummaryDNA replication of cultured fibroblasts of early passage derived from Werners syndrome (adult progeria) patients and from normal subjects were compared by DNA fiber autoradiography. The frequency of replication initiation was decreased in Werners syndrome cells derived from five patients compared with that in normal cells derived from three persons of different ages. The rate of DNA chain elongation did not differ between Werners syndrome cells and normal cells.

DNA polymerases η and θ function in the same genetic pathway to generate mutations at A/T during somatic hypermutation of Ig genes

Somatic hypermutation of the Ig genes requires the activity of multiple DNA polymerases to ultimately introduce mutations at both A/T and C/G base pairs. Mice deficient for DNA polymerase η (POLH) exhibited an ∼80% reduction of the mutations at A/T, whereas absence of polymerase θ (POLQ) resulted in ∼20% reduction of both A/T and C/G mutations. To investigate whether the residual A/T mutations observed in the absence of POLH are generated by POLQ and how these two polymerases might cooperate or compete with each other to generate A/T mutations, here we have established mice deficient for both POLH and POLQ. Polq–/–Polh–/– mice, however, did not show a further decrease of A/T mutations as compared with Polh–/– mice, suggesting that POLH and POLQ function in the same genetic pathway in the generation of these mutations. Frequent misincorporation of nucleotides, in particular opposite template T, is a known feature of POLH, but the efficiency of extension beyond the misincorporation differs significantly depending on the nature of the mispairing. Remarkably, we found that POLQ catalyzed extension more efficiently than POLH from all types of mispaired termini opposite A or T. Moreover, POLQ was able to extend mispaired termini generated by POLH albeit at a relatively low efficiency. These results reveal genetic and biochemical interactions between POLH and POLQ and suggest that POLQ might cooperate with POLH to generate some of the A/T mutations during the somatic hypermutation of Ig genes.

Prolongation of S phase and whole cell cycle in Werner's syndrome fibroblasts

Abstract The cell cycle was determined in early passage fibroblasts from Werners syndrome and normal subjects. The average cell cycle time was prolonged in Werners syndrome cells due to changes in the duration of S phase. Using alkaline sucrose gradient sedimentation, the rate of DNA elongation was examined, and no difference was observed between Werners syndrome cells and normal cells.