Dna2 is a multifunctional enzyme in yeast that possesses endonuclease activity well suited to remove RNA-DNA primers of Okazaki fragments, raising the question of whether endonuclease activity is essential for in vivo Dna2 function. Systematic site-directed mutations of amino acid residues in Saccharomyces cerevisiae DNA2 conserved in the central region of many eukaryotic DNA2 homologs allowed us to identify mutant dna2 alleles that were divided into three groups based on the viability of the mutant cells: (i) viable; (ii) inviable only when expression was repressed; (iii) inviable. Biochemical analyses of recombinant mutant Dna2 proteins isolated from the latter two groups revealed that they possessed normal ATPase/helicase activity, but were impaired in their endonuclease activity. Cells expressing mutant Dna2 enzymes partially impaired in endonuclease activity were viable, but were unable to grow when expression of their mutant Dna2 enzymes was further reduced. Their growth was restored when the mutant Dna2 proteins decreased in nuclease activity were induced to overexpress. In contrast, mutant Dna2 proteins lacking endonuclease activity did not allow cells to grow under any conditions tested. These in vivo and in vitro results demonstrate that the endonuclease activity of Dna2 is essential for Okazaki fragment processing.

Cardiomyocytes (CMs) derived from human pluripotent stem cells (hPSCs) are considered a most promising option for cell-based cardiac repair. Hence, various protocols have been developed for differentiating hPSCs into CMs. Despite remarkable improvement in the generation of hPSC-CMs, without purification, these protocols can only generate mixed cell populations including undifferentiated hPSCs or non-CMs, which may elicit adverse outcomes. Therefore, one of the major challenges for clinical use of hPSC-CMs is the development of efficient isolation techniques that allow enrichment of hPSC-CMs. In this review, we will discuss diverse strategies that have been developed to enrich hPSC-CMs. We will describe major characteristics of individual hPSC-CM purification methods including their scientific principles, advantages, limitations, and needed improvements. Development of a comprehensive system which can enrich hPSC-CMs will be ultimately useful for cell therapy for diseased hearts, human cardiac disease modeling, cardiac toxicity screening, and cardiac tissue engineering.

No abstract

Background:The biochemical function of variable N-terminal regions of eukaryotic Dna2 remains unclear. Results: The N-terminal 45-kDa domain of yeast Dna2 targets the enzyme specifically to a secondary structure flap. Conclusion:The hairpin binding activity of Dna2 contributes to efficient removal of hairpin flaps together with endonuclease and helicase activities during Okazaki fragment processing. Significance: The hairpin binding activity of Dna2 is critical for genome stability.

The Stra8 gene is a retinoic acid responsive gene. Its expression in premeiotic germ cells in both sexes implies that STRA8 protein plays roles for the progression of meiosis. Stra8 deficient germ cells do not initiate meiotic chromosome condensation and DNA double strand breaks. Its deficient mice were infertile. STRA8 is required for the transition into meiosis in both female and male germ cells. In addition its expression was confirmed in trophoblast giant cells in placenta, indicating another physiological role of STRA8 in other tissues. Yet it is still unclear how STRA8 is related in molecular level to meiosis and placentation. Here we report a possible function of STRA8 as a transcription factor. We have discovered that Stra8 gene is present in the nucleus and can activate basal transcription in F9 and P19 cells. Deletion mutant study revealed that C-terminus of STRA8 protein is important for its transcriptional activation activity. And N-terminus of the protein is critical for its nucleus localization. Moreover, random oligonucleotide electrophoresis mobility shift assay shows that the STRA8 directly binds to double strand DNA.