Akio Sugino

The phosphorylated C‐terminal domain of Xenopus Cut5 directly mediates ATR‐dependent activation of Chk1

ATR‐dependent activation of the kinase Chk1 is the initial step in signal transduction in the DNA replication checkpoint, which allows a cell to enter mitosis only after the completion of DNA replication. TopBP1‐related proteins in higher eukaryotes are implicated in the replication checkpoint, but their exact role remains elusive because of their requirements for replication initiation. Here we report that the initiation function of Xenopus Cut5/TopBP1 could be entirely separated from its checkpoint function: the N‐terminal half fragment, a region of Cut5 conserved through evolution, is sufficient for initiation, but is incapable of activating the checkpoint; the C‐terminal half fragment, which is unique in metazoan species, is by itself capable of activating the checkpoint response without initiating replication. Upon the activation of Chk1, the Ser1131 within the C‐terminal region of Cut5 is phosphorylated, and this phosphorylation is critical for the checkpoint response. Furthermore, Cut5 directly stimulated Chk1 phosphorylation in the in vitro kinase assay reconstituted with recombinant proteins and ATR immunoprecipitated from extracts. On the basis of replication protein A (RPA)‐dependent loading of Cut5 on to replicating and replication‐arrested chromatin, we propose that Cut5 plays a crucial role in the initial amplification step of the ATR‐Chk1 signaling pathway at the stalled replication fork.

Reconstitution of Saccharomyces cerevisiae prereplicative complex assembly in vitro

The assembly of the prereplicative complex (pre‐RC) at the origin of replication in eukaryotes is a highly regulated and highly conserved process that plays a critical role in preventing multiple rounds of DNA replication per cell division cycle. This study analyzes the molecular dynamics of the assembly of Saccharomyces cerevisiae pre‐RC in vitro using ARS1 plasmid DNA and yeast whole cell extracts. In addition, pre‐RC assembly was reconstituted in vitro using ARS1 DNA and purified origin‐recognition complex (ORC), Cdc6p and Cdt1p‐Mcm2‐7p. The results reveal sequential recruitment of ORC, Cdc6p, Cdt1p and Mcm2‐7p on to ARS1 DNA. When Mcm2‐7p is maximally loaded, Cdc6p and Cdt1p are released, suggesting that these two proteins are co‐ordinately regulated during pre‐RC assembly. In extracts from sid2‐21 mutant cells that are deficient in CDT1, ORC and Cdc6p bind to ARS1 but Cdt1p and Mcm2‐7p do not. However, Mcm2‐7p does bind in the presence of exogenous Cdt1p or Cdt1p‐Mcm2‐7p complex. Cdt1p‐Mcm2‐7p complex, which was purified from G1‐, early S or G2/M‐arrested cells, exhibits structure‐specific DNA binding, interacting only with bubble‐ or Y‐shape‐DNA, but the biological significance of this observation is not yet known.

Assembly of additional heterochromatin distinct from centromere-kinetochore chromatin is required for de novo formation of human artificial chromosome.

Alpha-satellite (alphoid) DNA is necessary for de novo formation of human artificial chromosomes (HACs) in human cultured cells. To investigate the relationship among centromeric, transcriptionally permissive and non-permissive chromatin assemblies on de novo HAC formation, we constructed bacterial artificial chromosome (BAC)-based linear HAC vectors whose left vector arms are occupied by βgeo coding genes with or without a functional promoter in addition to a common marker gene on the right arm. Although HACs were successfully generated from the vectors with promoter-less constructs on the left arm in HT1080 cells, we failed to generate a stable HAC from the vectors with a functional promoter on the left arm. Despite this failure in HAC formation, centromere components (CENP-A, CENP-B and CENP-C) assembled at the integration sites correlating with a transcriptionally active state of both marker genes on the vector arms. However, on the stable HAC, chromatin immunoprecipitation analysis showed that HP1α and trimethyl histone H3-K9 were enriched at the non-transcribing left vector arm. A transcriptionally active state on both vector arms is not compatible with heterochromatin formation on the introduced BAC DNA, suggesting that epigenetic assembly of heterochromatin is distinct from centromere chromatin assembly and is required for the establishment of a stable artificial chromosome.

Budding yeast mcm10/dna43 mutant requires a novel repair pathway for viability.

Background: MCM10 is essential for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae. Mcm10p functionally interacts with components of the pre‐replicative complex (Mcm2‐Mcm7 complex and origin recognition complex) as well as the pre‐initiation complex component (Cdc45p) suggesting that it may be a component of the pre‐RC as well as the pre‐IC. Two‐dimensional gel electrophoresis analysis showed that Mcm10p is required not only for the initiation of DNA synthesis at replication origins but also for the smooth passage of replication forks at origins. Genetic analysis showed that MCM10 interacts with components of the elongation machinery such as Polδ and Polɛ, suggesting that it may play a role in elongation replication.

Mechanism of DNA chain growth: VII. Direction and rate of growth of T4 nascent short DNA chains

Abstract A method has been developed for isolating the 5′-phosphoryl terminal portion of a DNA chain as a large oligonucleotide by successive digestion of DNA with spleen DNase II and spleen phosphodiesterase followed by gel filtration. Using this method, it was shown that after a 6- to 30-second pulse with [3H]thymidine at 8 °C, the label incorporated into completed or nearly completed T4 nascent short DNA chains (about 9 s) of both strands is not in their 5′-phosphoryl termini. However, if cells are labelled for one minute or longer, the label is found in the 5′-terminal portion of these short chains. A similar pattern emerges at 14 °C. At this temperature, the [3H]thymidine label begins to be found in the 5′-terminal region about ten seconds after addition of [3H]thymidine. Thus, the T4 nascent short DNA chains of both strands are synthesized in the 5′ → 3′ direction and the time required for the formation of a single short chain is about one minute at 8 °C and ten seconds at 14 °C. These conclusions are confirmed by another method, in which the 5′-terminal portion of a DNA chain is degraded selectively to mononucleotides by treatment with alkaline phosphatase, pancreatic DNase I and spleen phosphodiesterase.

Historical view of DNA replication studies, with special reference to Japan

Almost forty years after the key contributions to the field by Okazaki and coworkers that gave rise to the concept of leading and the lagging strand, we are still at the state of uncertainty about the proteins that replicate each strand. Perhaps, one main conclusion that should be drawn from the data currently available is that the protein architecture at the fork is more plastic than originally thought. iubmb Life, 58: 323‐327, 2006