::::::: My Cyber Lab(s) : ::::::
eIMBL Unit Labs
Faculty Members
DNA Replication
Systems Biology
Genomic Medicine
Molecular Oriental Medicine
Infectious Disease
DNA Replication
The progresses made during the past year on each of the proposed subjects by the members can be summarized as follows.
1) Genome-wide distribution of replication origins and replication machinery as well as early and late replicating segments
Masukata, in collaboration with Shirahige, conducted high-resolution mapping of replication initiation sites as well as the binding sites of replication factors on the entire chromosomes of fission yeast, and the results revealed intriguing facts about the distributions of potential origins and ORC/ MCM binding sites. Shirahige extended his high-density mapping analyses to sister chromatid cohesion proteins and recombination proteins to unravel the global dynamics of replication and recombination proteins during the cell cycle.
2) Molecular basis for selection of replication origins and determination of replication timing, as well as for their plasticity
Falaschi analyzed the role of DNA topoisomerases in the function of the lamin B2 replication origin. Masukata analyzed the mechanisms of the early and late replication timing on fission yeast replication origins. Masai proposed a role of MCM complex in selection of replication origins on the basis of its selective activation by single-stranded thymine-rich sequences. Masai, in collaboration with Shirahige, also searched for novel replication origins in the human and mouse cytokine cluster regions.
3) Assembly of initiation complexes and its activation for initiation of DNA synthesis
Araki conducted detailed analyses of assembly of initiation complexes at budding yeast replication origins. Masukata analyzed the order of assembly of replication factors at fission yeast replication origins, Masai and Lee conducted detailed analyses of Cdc7-mediated phosphorylation of MCM proteins, which may be relevant to the activation of preRC.
4) Architecture of the replication fork complex capable of efficient duplex unwinding and coordinated DNA synthesis
Shirahige analyzed the replication fork dynamics by monitoring the binding and movement of various replication fork factors using the high-density genomic tiling array. Tsurimoto biochemically dissected various clamp-clamp loader proteins which may function at the replication fork to coordinate various chromosome transactions. Hwang examined the functions of TopBP1 and MCM10 proteins in DNA replication in human cells using siRNA.
5) Cell cycle regulation of assembly, disassembly, degradation and activation of protein complexes required for the processes of DNA replication which permit once and only once DNA replication during a single cell cycle
Katayama conducted detailed biochemical and enzymatic analyses of regulation of origin firing at oriC of bacterial chromosomes using a reconstituted DNA replication system to elucidate the mechanisms of inhibition of re-replication through regulation of DnaA initiator. Hwang also conducted detailed analyses on how SeqA protein recognizes the hemimethylated DNA and its role in regulation of initiation at oriC. Araki analyzed the phosphorylation events and their target proteins, which take place at the onset of S phase. Takisawa used the Xenopus egg extract replication system to characterize the events taking place from pre-RC to pre-initiation complex (pre-IC) formation as well as the factors involved at the replication forks. Liang isolated a series of new budding yeast mutants which may affect the initiation of chromosomal replication in budding yeast and characterized them.
6) Cellular signal transduction pathways induced by perturbed replication forks
The roles of replication fork protection complex factors (Tof1-Csm3-Mrc1[yeast nomenclature]) were examined in yeast, Xenopus egg extracts and human cells (Takisawa, Shirahige and Masai). These studies revealed their conserved functions in checkpoint kinase activation in response to replication fork arrest. Tsurimoto conducted enzymatic characterization of checkpoint clamp-clamp loaders required for recognition of damages and replication fork blocks.
7) Machinery for processive DNA chain elongation and its modification during the course of fork inhibition
Seo conducted detailed analyses of processing of OKAZAKI fragment maturation which involves Flap endonuclease, DNA2 helicase and RPA.
8) Aberration of replication machinery and its regulators in diseases and during senescence
Fujita showed that the overexpression of Cdt1 can induce genetic instability in human cells, suggesting that the level of Cdt1 protein may be critically regulated to maintain the genetic integrity of the human cells. Masai showed that the reduction of Cdc7 protein level caused infertility in mice due to severe defect in development of germ cells.
9) Roles of replication factors in specific aspects of differentiation and development
Masai examined the expression profiles of various replication factors and cell cycle regulators in undifferentiated ES cells and compared them with those in differentiated cells, leading to identification of four critical factors which are highly overexpressed in undifferentiated ES cells and are quickly downregulated upon induction of differentiation.
10) Development of replication factors as novel therapeutic targets for prediction, diagnosis, treatment and prevention of diseases.
Liang showed that inhibition of replication factors, including Cdc6, Cdc45 and MCM, induced cell death specifically in cancer cells. Masai also made similar discovery with other replication factors.
