Haruhiko Takisawa

An auxin-based degron system for the rapid depletion of proteins in nonplant cells.

Plants have evolved a unique system in which the plant hormone auxin directly induces rapid degradation of the AUX/IAA family of transcription repressors by a specific form of the SCF E3 ubiquitin ligase. Other eukaryotes lack the auxin response but share the SCF degradation pathway, allowing us to transplant the auxin-inducible degron (AID) system into nonplant cells and use a small molecule to conditionally control protein stability. The AID system allowed rapid and reversible degradation of target proteins in response to auxin and enabled us to generate efficient conditional mutants of essential proteins in yeast as well as cell lines derived from chicken, mouse, hamster, monkey and human cells, thus offering a powerful tool to control protein expression and study protein function.

Identification of the yeast MCM3-related protein as a component of xenopus DNA replication licensing factor

Replication licensing factor is thought to be involved in the strict control of the initiation of DNA replication in eukaryotes. We identified a 100 kDa protein as a candidate for the licensing factor in Xenopus egg extracts. This protein was required for replication; it bound to sperm DNA before the formation of nuclei and apparently dissociated from the nuclear DNA during the progression of replication without being transported into the nuclei. An immunologically homologous protein in HeLa cells behaved similarly to the Xenopus protein during the cell cycle. Cloning and sequencing of the cDNAs encoding the Xenopus and human proteins revealed that they are homologs of yeast Mcm3, a putative yeast DNA replication licensing factor.

Xenopus Cdc45-dependent loading of DNA polymerase alpha onto chromatin under the control of S-phase Cdk.

At the onset of S phase, chromosomal replication is initiated by the loading of DNA polymerase α onto replication origins. However, the molecular mechanisms for controlling the initiation are poorly understood. Using Xenopus egg extract, we report here the identification of a Xenopus homolog of Cdc45, a yeast protein essential for the initiation of replication, which is shown to be an essential molecule for the initiation of replication via the loading of DNA polymerase α onto chromatin. XCdc45, by physically interacting with the polymerase in the extract, became associated with chromatin only after nuclear formation. During S phase, XCdc45 co‐localized with the polymerase in the nuclei, and the loading of the polymerase, which depended on endogenous XCdc45, was facilitated by exogenously added recombinant XCdc45. These findings, together with the apparent requirement of S‐phase‐cdk activity for the loading of XCdc45, suggest that XCdc45, under the control of S‐phase cdk, plays a pivotal role in the loading of DNA polymerase α onto chromatin.

Regulation of Replication Licensing by Acetyltransferase Hbo1

ABSTRACT The initiation of DNA replication is tightly regulated in eukaryotic cells to ensure that the genome is precisely duplicated once and only once per cell cycle. This is accomplished by controlling the assembly of a prereplicative complex (pre-RC) which involves the sequential binding to replication origins of the origin recognition complex (ORC), Cdc6/Cdc18, Cdt1, and the minichromosome maintenance complex (Mcm2-Mcm7, or Mcm2-7). Several mechanisms of pre-RC regulation are known, including ATP utilization, cyclin-dependent kinase levels, protein turnover, and Cdt1 binding by geminin. Histone acetylation may also affect the initiation of DNA replication, but at present neither the enzymes nor the steps involved are known. Here, we show that Hbo1, a member of the MYST histone acetyltransferase family, is a previously unrecognized positive regulatory factor for pre-RC assembly. When Hbo1 expression was inhibited in human cells, Mcm2-7 failed to associate with chromatin even though ORC and Cdc6 loading was normal. When Xenopus egg extracts were immunodepleted of Xenopus Hbo1 (XHbo1), chromatin binding of Mcm2-7 was lost, and DNA replication was abolished. The binding of Mcm2-7 to chromatin in XHbo1-depleted extracts could be restored by the addition of recombinant Cdt1.

The N-Terminal Noncatalytic Region of Xenopus RecQ4 Is Required for Chromatin Binding of DNA Polymerase α in the Initiation of DNA Replication

ABSTRACT Recruitment of DNA polymerases onto replication origins is a crucial step in the assembly of eukaryotic replication machinery. A previous study in budding yeast suggests that Dpb11 controls the recruitment of DNA polymerases α and ε onto the origins. Sld2 is an essential replication protein that interacts with Dpb11, but no metazoan homolog has yet been identified. We isolated Xenopus RecQ4 as a candidate Sld2 homolog. RecQ4 is a member of the metazoan RecQ helicase family, and its N-terminal region shows sequence similarity with Sld2. In Xenopus egg extracts, RecQ4 is essential for the initiation of DNA replication, in particular for chromatin binding of DNA polymerase α. An N-terminal fragment of RecQ4 devoid of the helicase domain could rescue the replication activity of RecQ4-depleted extracts, and antibody against the fragment inhibited DNA replication and chromatin binding of the polymerase. Further, N-terminal fragments of RecQ4 physically interacted with Cut5, a Xenopus homolog of Dpb11, and their ability to bind to Cut5 closely correlated with their ability to rescue the replication activity of the depleted extracts. Our data suggest that RecQ4 performs an essential role in the assembly of replication machinery through interaction with Cut5 in vertebrates.

The RLF-M component of the replication licensing system forms complexes containing all six MCM/P1 polypeptides

Replication licensing factor (RLF) is involved in preventing re‐replication of chromosomal DNA in a single cell cycle, and previously has been separated into two components termed RLF‐M and RLF‐B. Here we show that Xenopus RLF‐M consists of all six members of the MCM/P1 protein family, XMcm2–XMcm7. The six MCM/P1 polypeptides co‐eluted on glycerol gradients and gel filtration as complexes with a mol. wt of ∼400 kDa. In crude Xenopus extract, all six MCM/P1 polypeptides co‐precipitated with anti‐XMcm3 antibody, although only XMcm5 quantitatively co‐precipitated from purified RLF‐M. Further fractionation separated RLF‐M into two sub‐components, one consisting of XMcms 3 and 5, the other consisting of XMcms 2, 4, 6 and 7. Neither of the sub‐components provided RLF‐M activity. Finally, we show that all six MCM/P1 proteins bind synchronously to chromatin before the onset of S‐phase and are displaced as S–phase proceeds. These results strongly suggest that complexes containing all six MCM/P1 proteins are necessary for replication licensing.

Eukaryotic DNA replication: from pre-replication complex to initiation complex.

A common mechanism has emerged for the control of the initiation of eukaryotic DNA replication. The minichromosome maintenance protein complex (MCM) and Cdc45 have now been recognized as central components of the initiation machinery. In addition, two types of S phase promoting kinases conserved between yeast and humans play critical roles in the initiation reaction. At the onset of S phase, S phase kinases promote the association of Cdc45 with MCM at origins. Upon the formation of the MCM-Cdc45 complex at origins, the duplex DNA is unwound and various replication proteins, including DNA polymerases, are recruited onto unwound DNA. The increasing number of newly identified factors involved in the initiation reaction indicates that the control of initiation requires highly evolved machinery in eukaryotic cells.

Central role for cdc45 in establishing an initiation complex of DNA replication in Xenopus egg extracts.

In eukaryotes, chromosomal DNA is licensed to be replicated through the sequential loading of the origin recognition complex, Cdc6 and mini‐chromosome maintenance protein complex (MCM) onto chromatin. However, how the replication machinery is assembled onto the licensed chromatin during initiation of replication is poorly understood.

Xenopus Cut5 is essential for a CDK‐dependent process in the initiation of DNA replication

Fission yeast Cut5/Rad4 and its budding yeast homolog Dpb11 are required for both DNA replication and the S‐phase checkpoint. Here, we have investigated the role of the Xenopus homolog of Cut5 in the initiation of DNA replication using Xenopus egg extracts. Xenopus Cut5, which shows sequence similarity to DmMus101 and HsTopBP1, is essential for DNA replication in the egg extracts. It is required for the chromatin binding of Cdc45 and DNA polymerases, but not for the formation of pre‐replicative complexes or the elongation stage of DNA replication. The chromatin binding of Cut5 consists of two distinct modes. S‐phase cyclin‐dependent kinase (S‐CDK)‐independent binding is sufficient for DNA replication while S‐CDK‐dependent binding is dispensable. Further, S‐CDK acts after the chromatin binding of Cut5 and before the binding of Cdc45. These results demonstrate that the chromatin binding of Cut5 is required for the action of S‐CDK, which in turn triggers the formation of pre‐initiation complexes of DNA replication.

Licensing of DNA replication by a multi-protein complex of MCM/P1 proteins in Xenopus eggs

In eukaryotes, chromosomal DNA is licensed for a single round of replication in each cell cycle. Xenopus MCM3 protein has been implicated in the licensing of replication in egg extract. We have cloned cDNAs encoding five immunologically distinct proteins associated with Xenopus MCM3 as members of the MCM/P1 family. Six Xenopus MCM proteins formed a physical complex in the egg extract, bound to unreplicated chromatin before the formation of nuclei, and apparently displaced from replicated chromatin. The requirement of six XMCM proteins for the replication activity of the egg extract before nuclear formation suggests that their re‐association with replicated chromatin at the end of the mitotic cell cycle is a key step for the licensing of replication.