Cdc7 kinase, conserved from yeasts to human, plays important roles in DNA replication. However, the mechanisms by which it stimulates initiation of DNA replication remain largely unclear. We have analyzed phosphorylation of MCM subunits during cell cycle by examining mobility shift on SDS-PAGE. MCM4 on the chromatin undergoes specific phosphorylation during S phase. Cdc7 phosphorylates MCM4 in the MCM complexes as well as the MCM4 N-terminal polypeptide. Experiments with phospho-amino acid-specific antibodies indicate that the S phase-specific mobility shift is due to the phosphorylation at specific N-terminal (S/T)(S/T)P residues of the MCM4 protein. These specific phosphorylation events are not observed in mouse ES cells deficient in Cdc7 or are reduced in the cells treated with siRNA specific to Cdc7, suggesting that they are mediated by Cdc7 kinase. The N-terminal phosphorylation of MCM4 stimulates association of Cdc45 with the chromatin, suggesting that it may be an important phosphorylation event by Cdc7 for activation of replication origins. Deletion of the N-terminal non-conserved 150 amino acids of MCM4 results in growth inhibition, and addition of amino acids carrying putative Cdc7 target sequences partially restores the growth. Furthermore, combination of MCM4 N-terminal deletion with alanine substitution and deletion of the N-terminal segments of MCM2 and MCM6, respectively, which contain clusters of serine/threonine and are also likely targets of Cdc7, led to an apparent nonviable phenotype. These results are consistent with the notion that the N-terminal phosphorylation of MCM2, MCM4, and MCM6 may play functionally redundant but essential roles in initiation of DNA replication.

We generated self-induced retinal ganglion cells (RGCs) with functional axons from human induced pluripotent stem cells. After development of the optic vesicle from the induced stem cell embryoid body in three-dimensional culture, conversion to two-dimensional culture, achieved by supplementation with BDNF, resulted in differentiation of RGCs at a rate of nearly 90% as indicated by a marginal subregion of an extruded clump of cells, suggesting the formation of an optic vesicle. Axons extended radially from the margin of the clump. Induced RGCs expressed specific markers, such as Brn3b and Math5, as assessed using by quantitative PCR and immunohistochemistry. The long, prominent axons contained neurofilaments and tau and exhibited anterograde axonal transport and sodium-dependent action potentials. The ability to generate RGCs with functional axons uniformly and at a high rate may contribute to both basic and clinical science, including embryology, neurology, pathognomy, and treatment of various optic nerve diseases that threaten vision.

No abstract

In eubacteria, PriA helicase detects the stalled DNA replication forks. This critical role of PriA is ascribed to its ability to bind to the 3 0 end of a nascent leading DNA strand in the stalled replication forks. The crystal structures in complexes with oligonucleotides and the combination of fluorescence correlation spectroscopy and mutagenesis reveal that the N-terminal domain of PriA possesses a binding pocket for the 3 0 -terminal nucleotide residue of DNA. The interaction with the deoxyribose 3 0 -OH is essential for the 3 0 -terminal recognition. In contrast, the direct interaction with 3 0 -end nucleobase is unexpected, considering the same affinity for oligonucleotides carrying the four bases at the 3 0 end. Thus, the N-terminal domain of PriA recognizes the 3 0 -end base in a base-non-selective manner, in addition to the deoxyribose and 5 0 -side phosphodiester group, of the 3 0 -terminal nucleotide to acquire both sufficient affinity and non-selectivity to find all of the stalled replication forks generated during DNA duplication. This unique feature is prerequisite for the proper positioning of the helicase domain of PriA on the unreplicated double-stranded DNA.

Arrest of replication forks by various internal and external threats evokes a myriad of cellular reactions, collectively known as DNA replication checkpoint responses. In bacteria, PriA is essential for restoration of stalled replication forks and recombinational repair of double-stranded DNA breaks and is a candidate sensor protein that may recognize arrested forks. Here, we report that PriA protein specifically recognizes 3 termini of arrested nascent DNA chains at model stalled replication forks in vitro. Mutations in the putative "3 terminus binding pocket" present in the N-terminal segment of PriA result in reduced binding to stalled replication fork structures and loss of its biological functions. The results suggest a mechanism by which stalled replication forks are recognized by a sensor protein for checkpoint responses.Progression of replication forks is stalled by a variety of internal and external causes, including DNA damage and depletion of precursors for DNA synthesis. The arrested replication forks elicit checkpoint responses that enable cells to repair damage, restore replication forks, and restart DNA replication (1, 2). A number of eukaryotic gene products have been identified that participate in this process. The initial phase of this response is the recognition of the arrested forks by a sensor protein, which may be recruited to the site of the fork arrest and transduce various signals for further downstream cellular responses. However, the nature of the protein that detects the stalled replication forks and the mechanism by which they are recognized are still unclear.In Escherichia coli, PriA, a DEXH-type DNA helicase originally discovered as a protein essential for replication of a small single-stranded phage DNA, X174, is believed to play a key role in rescuing the stalled replication forks (1, 3-5). The priA null cells display numerous phenotypes, including low viability and sensitivity to DNA damaging agents such as UV, ␥-ray, and mitomycin C (6 -10). These defects may reflect a critical role played by PriA in resumption of DNA replication after arrest of ongoing replication forks (11). In E. coli, in fact, arrest of ongoing replication forks induces a recombination-dependent mode of DNA replication, known as inducible or constitutive stable DNA replication (iSDR 1 or cSDR, respectively), which requires PriA protein (8). It was shown that the X174-type primosome could be assembled on a model D-loop structure in vitro (12, 13). It has been proposed that PriA may recognize recombination intermediates generated as a result of fork arrest as well as the arrested replication fork per se and facilitate reassembly of replication forks (8,14,15). Consistent with this, PriA recognizes and binds specifically to DNA structures such as those mimicking D-loop (intermediates of homologous recombination reactions) or arrested replication forks (16 -18). However, it has not been known what structural features of D-loop or arrested DNA replication fork structures are recognized by PriA. In this report, w...