Advances in Biophysics最新文献

Bloom syndrome is a rare autosomal recessive genetic disorder characterized by lupus-like erythematous telangiectasias of the face, sun sensitivity, stunted growth, and immunodeficiency. Chromosome instability syndromes have a common feature, being associated at high frequency with neoplasia. BS is considered as one of the chromosome instability syndromes since the fibroblasts or lymphocytes of BS patients show excessive spontaneous chromosome instability. The causative gene of BS (BLM) was identified as a RecQ helicase homologue. In this review, we showed the characteristic phenotypes of BS, especially two Japanese siblings. In the latter of the review, the functional domains of BLM, those are nuclear localization signal and the interacting proteins such as ATM, are shown. Several lines of reports indicates that BLM helicase is involved in the re-initiation of DNA replication at sites where replication forks have arrested or collapsed. To elucidate the precise function of RecQ helicase in DNA repair and replication aims not only to improve our understanding of the molecular basis for tumorigenesis, but also to extend the range of potential therapeutic targets.

Pairing between homologous DNA controls cellular functions including double-strand break repair, mitotic recombination, and progression of DNA replication forks, as well as chiasma formation during meiosis. Here I summarize that homologous interaction could promote the cell killing in bacteria, yeast, and multicellular organisms. The mechanisms of cell killing are categorized into two types: (1) the killing due to the accumulation of extrachromosomal DNA; (2) the killing induced by Holliday junction structures. I propose that the mechanisms of such killing function as novel apoptotic pathways in the cells carrying severe DNA damages to eliminate such damages from cell population.

Repair of DSBs is important to prevent chromosomal fragmentation, translocations and deletions. To investigate the process in NHEJ, we have established an in vitro system to clarify the measurement and analysis of the efficiency and the fidelity of rejoining of DSBs, and applied the method to investigate NHEJ in human cells derived from patients suffering from cancer-prone hereditary diseases. A DSB was introduced in plasmid pZErO-2 at a specific site within the ccdB gene that is lethal to E. coli cells, and treated with nuclear extracts from human cells. The efficiency of rejoining in the nuclear extract from an A-T cell line was comparable to that from a control cell line. However, the accuracy of rejoining was much lower for the A-T cell extract than for the control cell extract. All mutations were deletions, most of which contained short direct repeats at the breakpoint junctions. The deletion spectrum caused by the A-T nuclear extract was distinct from that by the control extract. These results indicate that A-T cells have certain deficiencies in end-joining of double-strand breaks in DNA. The extract from BS cells also showed the similar activity and the lower fidelity of rejoing compared to that from normal cells. From the sequencing analysis of the junction of DSBs, it is speculated that the defect in the BLM helicase might cause irregular rejoining of DSBs. Radioadaptive response is the acquirement of cellular resistance to ionizing radiation by prior exposure to low dose. We investigated the in vitro end-joining activity of DNA ends in radioadaptive cells. Both the efficiency and the fidelity of rejoining in the cells pre-exposed to low dose are increased comparing to those without pre-exposure. We also investigated the joining activity of DNA ends in p53-deficient cells. Pre-irradiation caused no apparent alteration in both the efficiency and fidelity of end-joining. These results suggest that the exposure to low dose activates a cellular function to repair DSBs efficiently, which is dependent on p53. These results indicate that NHEJ pathway is regulated by many factors; genetic regulation by ATM and BLM, and physiological conditions such as irradiation with ionizing radiation. The observations also suggest that in some occasions p53 might play a key role in NHEJ.

In Saccharomyces cerevisiae, VMAI intein encodes a homing endonuclease termed VDE which is produced by an autocatalytic protein splicing reaction. VDE introduces a DSB at its recognition sequence on intein-minus allele, resulting in the lateral transfer of VMAI intein. In this review, we summarize a decade of in vitro study on VDE and describe our recent study on the in vivo behavior of both VDE and host proteins involved in intein mobility. Meiotic DSBs caused by VDE are repaired in the similar pathway to that working in meiotic recombination induced by Spollp-mediated DSBs. Meiosis-specific DNA cleavage and homing is shown to be guaranteed by the two distinct mechanisms, the subcellular localization of VDE and a requirement of premeiotic DNA replication. Based on these lines of evidence, we present the whole picture of molecular mechanism of VDEinitiated homing in yeast cells.

Repair of DSBs is important to prevent chromosomal fragmentation, translocations and deletions. To investigate the process in NHEJ, we have established an in vitro system to clarify the measurement and analysis of the efficiency and the fidelity of rejoining of DSBs, and applied the method to investigate NHEJ in human cells derived from patients suffering from cancer-prone hereditary diseases. A DSB was introduced in plasmid pZErO-2 at a specific site within the ccdB gene that is lethal to E. coli cells, and treated with nuclear extracts from human cells. The efficiency of rejoining in the nuclear extract from an A-T cell line was comparable to that from a control cell line. However, the accuracy of rejoining was much lower for the A-T cell extract than for the control cell extract. All mutations were deletions, most of which contained short direct repeats at the breakpoint junctions. The deletion spectrum caused by the A-T nuclear extract was distinct from that by the control extract. These results indicate that A-T cells have certain deficiencies in end-joining of double-strand breaks in DNA. The extract from BS cells also showed the similar activity and the lower fidelity of rejoing compared to that from normal cells. From the sequencing analysis of the junction of DSBs, it is speculated that the defect in the BLM helicase might cause irregular rejoining of DSBs. Radioadaptive response is the acquirement of cellular resistance to ionizing radiation by prior exposure to low dose. We investigated the in vitro end-joining activity of DNA ends in radioadaptive cells. Both the efficiency and the fidelity of rejoining in the cells pre-exposed to low dose are increased comparing to those without pre-exposure. We also investigated the joining activity of DNA ends in p53-deficient cells. Pre-irradiation caused no apparent alteration in both the efficiency and fidelity of end-joining. These results suggest that the exposure to low dose activates a cellular function to repair DSBs efficiently, which is dependent on p53. These results indicate that NHEJ pathway is regulated by many factors; genetic regulation by ATM and BLM, and physiological conditions such as irradiation with ionizing radiation. The observations also suggest that in some occasions p53 might play a key role in NHEJ.

The Tol2 element of the medaka fish is a member of the hAT (hobo/Activator/ Tam3) transposable element family. About 20 copies are present in the medaka fish genome and, unlike many other hAT family elements, virtually all the copies are autonomous or potentially autonomous, containing an intact transposase gene. Excision of Tol2 is not precise at the nucleotide sequence level, excision foot-prints being heterogeneous. In more than half of excision events, however, breakage and rejoining of DNA molecules occur within the 8-bp target site duplication region, removing the entire Tol2 sequence and retaining parts of the target site duplications. In the reminder of the excision events, either the left or the right terminal region is left and the other end is lost together with its flanking region. Thus, there might be two different mechanisms of excision. Insertion of Tol2 occurs without detectable preference for target sequences and creates a target site duplication of exactly 8 bp. In addition to the medaka fish and related fish species, Tol2 transposes in mammalian cells in culture, including human and mouse examples. Autonomy is also retained in these cases. A gene transfer vector using Tol2 has already been established in fish. Foreign DNA fragements inserted in Tol2 can be efficiently delivered to the chromosomes by transposition. The latest version of the vector contains, between the Tol2 terminal regions, a bacterial drug-resistance gene and a plasmid replication origin. This allows simple recovery of insertion regions, as plasmid DNA, from genomic DNA of transformants. Modification of this system for other vertebrates, especially for mammals, are now in progress.

The isolation of the NBS1 gene revealed the molecular mechanisms of DSB repair. In response to DNA damage, histone H2AX in the vicinity of DSBs is phosphorylated by ATM. NBS1 then targets the MRE11/RAD50 complex to the sites of DSBs through interaction of the FHA/BRCT domain with gamma-H2AX. NBSI complex binds to damaged-DNA directly, and HR repair is initiated. To collaborate DSB repair, ATM also regulates cell cycle checkpoints at GI, G2, and intra-S phases via phosphorylation of SMC, CHK2 and FANCD2. The phosphorylation of these proteins require NBS1 complex. Thus, NBSI has at least two important roles in genome maintenance, as a DNA repair protein in HR pathway and as a signal modifier in intra-S phase checkpoints. NBSI is also known to be involved in maintenance of telomores, which have DSB-like structures and defects here can cause telomcric fusion. Therefore, NBS1 should be a multi-functional protein for the maintenance of genomic integrity. Further studies on NBS1 will provide insights into the mechanisms of DNA damage response and the network of these factors involved in genomic stability.