A Second Human Dbf4/ASK-related Protein, Drf1/ASKL1, Is Required for Efficient Progression of S and M Phases

Abstract

Cdc7-Dbf4 kinase is conserved through evolution and regulates initiation and progression of DNA replication. In human, ASK/hsDbf4 binds and activates huCdc7 during S phase and this kinase complex is essential for DNA replication and cell proliferation. Drf1/ASKL1, a second human Dbf4/ASK-related protein, shares three conserved Dbf4 motifs previously identified on all of the Dbf4-related molecules. Drf1/ASKL1 can bind and activate huCdc7, and Cdc7-ASKL1 complex phosphorylates MCM2. ASKL1 transcription and protein levels oscillate during cell cycle and increase at late S to G 2 /M phases. The protein is detected predominantly in the nuclearsoluble fraction but not in the chromatin-bound fraction. Inhibition of Drf1/ASKL1 expression by siRNA results in attenuation of cell growth and in the increase of late S and G 2 /M phase population. siRNA treatment on synchronized cell population revealed that S phase progression is delayed when ASKL1 protein level is decreased. S phase delay may be linked to replication fork block, because increased levels of ␥H2AX and activated form of Chk2 are detected with ASKL1 siRNA in the absence of any additional DNA damages. Furthermore, mitotic progression is retarded in ASKL1 or Cdc7 siRNA-treated cells. Our results suggest that ASKL1 in a complex with Cdc7 may play a role in normal progression of both S and M phases.Cell cycle progression of eukaryotic cells is strictly regulated by a series of phosphorylation events. Among them, multiple cyclin-dependent kinases play crucial roles to facilitate the progression of cell cycle at various stages. Multiple cyclins and cyclin-dependent kinases have been identified in various eukaryotes, each acting at specific stages of the cell cycles (1-7). Conserved cyclin-box sequences have been identified on the cyclin molecules, and significant homology is present in the kinase-conserved domains of various cyclin-dependent kinases (8, 9).Cdc7-Dbf4 is another family of serine-threonine kinases originally identified in Saccharomyces cerevisiae (10,11). Recent studies have shown conservation of Cdc7-Dbf4 kinases throughout evolution and their essential roles in DNA replication in higher eukaryotes (12-23). In fission yeast, the presence of a second set of Cdc7-Dbf4 kinase, Spo4-Spo6, was reported (24, 25), whereas only one set of Cdc7-Dbf4 has been identified on the genome of budding yeast. Spo4 and Spo6 were shown to play essential roles during meiosis but not during the mitotic cell cycle, in contrast to Hsk1-Dfp1/Him1, which is essential for cell viability. The presence of more than one Cdc7-Dbf4-related kinase in a single eukaryotic species suggests a possibility that they also constitute a novel kinase family, each member of which may play distinct roles in cell cycle progression.We have identified a novel human cDNA, designated ASKL1, 1 which shares significant homology with Dbf4 protein family. ASKL1 is identical to Drf1 reported recently by Montagnoli et al. (26). It was reported that Drf1 binds to huCdc7 and activates its kinase activity and ...

Xenopus CDC7/DRF1 Complex Is Required for the Initiation of DNA Replication

“…Indeed, our probes for Northern and Western analysis were directed against the C-terminal half of the coding region that is the least conserved region of the protein between species (8% identity between Xenopus and Human Drf1). The identification of Drf1 in various human and mouse tissues, as well as cell lines, supports the idea that a zygotic form of XDrf1 exists in late Xenopus embryos and somatic cells (8,10). Contrary to XDrf1, our data suggest that there is only one XDbf4 gene that is expressed throughout development.…”

“…This complex is often referred to as DDK (Dbf4-dependent kinase). The existence in fission yeast of a Cdc7/Dbf4 complex paralog, Spo4/Spo6, and the recent discovery of multiple Dbf4-related molecules in animal cells suggest that they belong to a novel DDK protein kinase family (7)(8)(9)(10). Functional differences between family members have started to emerge but they need to be further characterized.…”

“…Two DDKs have recently been identified in humans and Xenopus laevis, the Cdc7/Dbf4 and Cdc7/Drf1 complexes (9,13,14). Whereas both kinase complexes are able to phosphorylate the Mcm2 protein in vitro, the Cdc7/Dbf4 complex is believed to be the one essential for DNA replication (10,13,14). On the other hand, the Cdc7/Drf1 complex appears to be required for efficient progression through S and M phases in human but is dispensable for initiation of DNA replication in the cell-free system derived from Xenopus eggs (9,10).…”

“…Whereas both kinase complexes are able to phosphorylate the Mcm2 protein in vitro, the Cdc7/Dbf4 complex is believed to be the one essential for DNA replication (10,13,14). On the other hand, the Cdc7/Drf1 complex appears to be required for efficient progression through S and M phases in human but is dispensable for initiation of DNA replication in the cell-free system derived from Xenopus eggs (9,10). In addition, both Xenopus complexes seem to play a role in checkpoint pathways.…”

Hsk1 kinase is required for induction of meiotic dsDNA breaks without involving checkpoint kinases in fission yeast

“…Therefore, the block of meiosis I in hsk1-89 cells may not be simply caused by the absence of recombination. In mammalian cells, Cdc7 has been implicated in mitosis as well (38). Therefore, we speculate that Hsk1 may play some unknown essential functions during meiotic nuclear and cell divisions independent from its S-phase functions.…”