Mitsuyoshi Yamazoe

Cell Cycle–Dependent Duplication and Bidirectional Migration of SeqA-Associated DNA–Protein Complexes in E. coli

Using immunofluorescence microscopy, we have found that SeqA protein, a regulator of replication initiation, is localized as discrete fluorescent foci in E. coli wild-type cells. Surprisingly, SeqA foci were observed also in an oriC deletion mutant. Statistical analysis revealed that a SeqA focus is localized at midcell in newborn cells. The SeqA focus is duplicated and tethered at midcell until an FtsZ ring is formed. Subsequently, these foci migrate in opposite directions toward cell quarter sites and remain tethered there until the cell divides. The cell cycle-dependent bidirectional migration of SeqA-DNA complexes is quite different from the migration pattern of oriC Dna copies. MukB protein is required for correct localization of SeqA complexes by an unknown mechanism.

Multiple Repair Pathways Mediate Tolerance to Chemotherapeutic Cross-linking Agents in Vertebrate Cells

Cross-linking agents that induce DNA interstrand cross-links (ICL) are widely used in anticancer chemotherapy. Yeast genetic studies show that nucleotide excision repair (NER), Rad6/Rad18-dependent postreplication repair, homologous recombination, and cell cycle checkpoint pathway are involved in ICL repair. To study the contribution of DNA damage response pathways in tolerance to cross-linking agents in vertebrates, we made a panel of gene-disrupted clones from chicken DT40 cells, each defective in a particular DNA repair or checkpoint pathway, and measured the sensitivities to cross-linking agents, including cis-diamminedichloroplatinum (II) (cisplatin), mitomycin C, and melphalan. We found that cells harboring defects in translesion DNA synthesis (TLS), Fanconi anemia complementation groups (FANC), or homologous recombination displayed marked hypersensitivity to all the cross-linking agents, whereas NER seemed to play only a minor role. This effect of replication-dependent repair pathways is distinctively different from the situation in yeast, where NER seems to play a major role in dealing with ICL. Cells deficient in Rev3, the catalytic subunit of TLS polymerase Polzeta, showed the highest sensitivity to cisplatin followed by fanc-c. Furthermore, epistasis analysis revealed that these two mutants work in the same pathway. Our genetic comprehensive study reveals a critical role for DNA repair pathways that release DNA replication block at ICLs in cellular tolerance to cross-linking agents and could be directly exploited in designing an effective chemotherapy.

Complex formation of MukB, MukE and MukF proteins involved in chromosome partitioning in Escherichia coli

mukF, mukE and mukB genes are essential for the process of chromosome partitioning in Escherichia coli. We have studied protein–protein interactions among MukB, MukE and MukF proteins by co‐immunoprecipitation and sucrose gradient sedimentation experiments, using mukFEB null cells harboring plasmids carrying the wild‐type or mutant‐type mukFEB operon. MukB forms a complex with MukF and MukE. Analysis of mutant MukB proteins suggested that MukF and MukE bind the C‐terminal globular domain of MukB. MukF is indispensable for an interaction between MukB and MukE; however, MukF itself is able to associate with MukB even in the absence of MukE. We have also found that MukF has a Ca2+‐binding activity. Although purified MukF was able to make a complex either with MukE or MukB, a complex consisting of the three Muk proteins was barely detected in vitro. However, increasing the Ca2+ or Mg2+ concentration in the reaction partially restored complex formation. This suggests that Ca2+ or Mg2+ may be required for the formation of a complex consisting of the three Muk proteins, and thus may participate in a particular step during chromosome partitioning.

Cells Deficient in the FANC/BRCA Pathway Are Hypersensitive to Plasma Levels of Formaldehyde

Formaldehyde is an aliphatic monoaldehyde and is a highly reactive environmental human carcinogen. Whereas humans are continuously exposed to exogenous formaldehyde, this reactive aldehyde is a naturally occurring biological compound that is present in human plasma at concentrations ranging from 13 to 97 micromol/L. It has been well documented that DNA-protein crosslinks (DPC) likely play an important role with regard to the genotoxicity and carcinogenicity of formaldehyde. However, little is known about which DNA damage response pathways are essential for cells to counteract formaldehyde. In the present study, we first assessed the DNA damage response to plasma levels of formaldehyde using chicken DT40 cells with targeted mutations in various DNA repair genes. Here, we show that the hypersensitivity to formaldehyde is detected in DT40 mutants deficient in the BRCA/FANC pathway, homologous recombination, or translesion DNA synthesis. In addition, FANCD2-deficient DT40 cells are hypersensitive to acetaldehyde, but not to acrolein, crotonaldehyde, glyoxal, and methylglyoxal. Human cells deficient in FANCC and FANCG are also hypersensitive to plasma levels of formaldehyde. These results indicate that the BRCA/FANC pathway is essential to counteract DPCs caused by aliphatic monoaldehydes. Based on the results obtained in the present study, we are currently proposing that endogenous formaldehyde might have an effect on highly proliferating cells, such as bone marrow cells, as well as an etiology of cancer in Fanconi anemia patients.

Bidirectional migration of SeqA‐bound hemimethylated DNA clusters and pairing of oriC copies in Escherichia coli

We previously found that SeqA protein, which binds preferentially to newly replicated hemimethylated DNA, is localized as discrete fluorescent foci in Escherichia coli cells. A single SeqA focus, localized at midcell, separates into two foci and these foci migrate abruptly in opposite directions.

RAD51 Up-regulation Bypasses BRCA1 Function and Is a Common Feature of BRCA1-Deficient Breast Tumors

The breast cancer susceptibility gene BRCA1 encodes a large protein thought to contribute to a variety of cellular processes, although the critical determinants of BRCA1-deficient tumorigenesis remain unclear. Given that BRCA1 is required for cell proliferation, suppressor mutations are believed to modify BRCA1 phenotypes and contribute to the etiology of BRCA1-deficient tumors. Here, we show that overexpression of the homologous recombinase RAD51 in a DT40 BRCA1Delta/Delta mutant rescues defects in proliferation, DNA damage survival, and homologous recombination (HR). In addition, epistasis analysis with BRCA1 and the DNA end-joining factor KU70 indicates that these factors operate independently of one another to repair double-strand breaks. Consistent with this genetic finding, cell synchronization studies show that the ability of BRCA1 to promote radioresistance is restricted to the late S and G2 phases of the cell cycle, as predicted for genes whose function is specific to homology-mediated repair rather than nonhomologous end-joining. Notably, retrospective analyses of microarray expression data reveal elevated expression of RAD51 and two of its late-acting cofactors, RAD54 and RAD51AP1, in BRCA1-deficient versus sporadic breast tumors. Taken together, our results indicate that up-regulation of HR provides a permissive genetic context for cells lacking BRCA1 function by circumventing its requirement in RAD51 subnuclear assembly. Furthermore, the data support a model in which enhanced HR activity contributes to the etiology of BRCA1-deficient tumors.

Functional relationships of FANCC to homologous recombination, translesion synthesis, and BLM

Some of the restarting events of stalled replication forks lead to sister chromatid exchange (SCE) as a result of homologous recombination (HR) repair with crossing over. The rate of SCE is elevated by the loss of BLM helicase or by a defect in translesion synthesis (TLS). We found that spontaneous SCE levels were elevated ∼2‐fold in chicken DT40 cells deficient in Fanconi anemia (FA) gene FANCC. To investigate the mechanism of the elevated SCE, we deleted FANCC in cells lacking Rad51 paralog XRCC3, TLS factor RAD18, or BLM. The increased SCE in fancc cells required Xrcc3, whereas the fancc/rad18 double mutant exhibited higher SCE than either single mutant. Unexpectedly, SCE in the fancc/blm mutant was similar to that in blm cells, indicating functional linkage between FANCC and BLM. Furthermore, MMC‐induced formation of GFP‐BLM nuclear foci was severely compromised in both human and chicken fancc or fancd2 cells. Our cell survival data suggest that the FA proteins serve to facilitate HR, but not global TLS, during crosslink repair.

Generation of medaka gene knockout models by target-selected mutagenesis

We have established a reverse genetics approach for the routine generation of medaka (Oryzias latipes) gene knockouts. A cryopreserved library of N-ethyl-N-nitrosourea (ENU) mutagenized fish was screened by high-throughput resequencing for induced point mutations. Nonsense and splice site mutations were retrieved for the Blm, Sirt1, Parkin and p53 genes and functional characterization of p53 mutants indicated a complete knockout of p53 function. The current cryopreserved resource is expected to contain knockouts for most medaka genes.

Distribution of neuropeptide Y in the lower brainstem: an immunohistochemical analysis

The distribution of neuropeptide Y (NPY)-like immunoreactive (NPYI) structures in the rat lower brainstem was examined by means of indirect immunofluorescence or peroxidase-antiperoxidase methods. In addition to the well known immunoreactive NPYI containing cell groups, the present study demonstrated a much wider distribution of immunoreactive NPYI cells in the lower brainstem, i.e. substantia nigra pars lateralis, interpeduncular nucleus, inferior colliculus, nucleus cuneiformis, dorsal tegmental nucleus of Gudden, nucleus laterodorsalis tegmenti, nucleus vestibularis medialis, nucleus vestibularis inferioris, in the fasciculus longitudinalis medialis and nucleus parvocellularis compacta. We also demonstrated an extensive network of NPYI fibers in various areas of the lower brainstem including the auditory system, viscerosensory system, visceromotor system, raphe nuclei, reticular formation, parabrachial area, locus coeruleus and interpeduncular nucleus, etc. The possible importance of NPY is briefly discussed.

The assembly and migration of SeqA–Gfp fusion in living cells of Escherichia coli

SeqA protein, which binds to hemi‐methylated GATC sequences of DNA, is localized to discrete fluorescent foci in wild‐type Escherichia coli cells. In this work, we observed cellular localization of the SeqA–Gfp fusion in living cells. SeqA–Gfp was localized to a discrete focus or foci in wild‐type and seqA null mutant cells, but the fusion was dispersed in the whole cell in dam null mutant cells lacking Dam methyltransferase. These results were consistent with the previous description of the localization of SeqA by immunofluorescence microscopy. Time‐lapse experiments revealed that duplicated SeqA–Gfp foci migrated rapidly in opposite directions. Flow cytometry demonstrated that the fusion restored synchronous replication of chromosomal DNA from multiple origins in seqA null mutant cells, indicating that SeqA–Gfp is biologically active. Immunoprecipitation of the fusion from cell extracts using anti‐Gfp antibody indicated that the fusion was assembled with the wild‐type SeqA protein.