Search for apoptotic nucleases in yeast: role of Tat-D nuclease in apoptotic DNA degradation.
DNA fragmentation/degradation is an important step for apoptosis. However, in unicellular organisms such as yeast, this process has rarely been investigated. In the current study, we revealed eight apoptotic nuclease candidates in Saccharyomyces cerevisiae, analogous to the Caenorhabditis elegans apoptotic nucleases. One of them is Tat-D. Sequence comparison indicates that Tat-D is conserved across kingdoms, implicating that it is evolutionarily and functionally indispensable. In order to better understand the biochemical and biological functions of Tat-D, we have overexpressed, purified, and characterized the S. cerevisiae Tat-D (scTat-D). Our biochemical assays revealed that scTat-D is an endo-/exonuclease. It incises the double-stranded DNA without obvious specificity via its endonuclease activity and excises the DNA from the 3'- to 5'-end by its exonuclease activity. The enzyme activities are metal-dependent with Mg(2+) as an optimal metal ion and an optimal pH around 5. We have also identified three amino acid residues, His(185), Asp(325), and Glu(327), important for its catalysis. In addition, our study demonstrated that knock-out of TAT-D in S. cerevisiae increases the TUNEL-positive cells and cell survival in response to hydrogen hyperoxide treatment, whereas overexpression of Tat-D facilitates cell death. These results suggest a role of Tat-D in yeast apoptosis.
Qiu
Junzhuan
J
Department of Radiation Biology, City of Hope National Medical Center and Beckman Research Institute, Duarte, California 91010, USA. jqiu@coh.org
Yoon
Jung-Hoon
JH
Shen
Binghui
B
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D017173:Caenorhabditis elegans; D002470:Cell Survival; D004247:DNA / Q000378:metabolism; D053938:DNA Fragmentation; D019992:Databases as Topic; D004706:Endodeoxyribonucleases / Q000096:biosynthesis / Q000502:physiology*; D004720:Endonucleases / Q000737:chemistry; D005090:Exodeoxyribonucleases / Q000096:biosynthesis / Q000502:physiology*; D005092:Exonucleases / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry*; D020287:In Situ Nick-End Labeling; D008274:Magnesium / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D010802:Phylogeny; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D005656:Fungal Proteins; D009841:Oligonucleotides; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D006861:Hydrogen Peroxide; D006867:Hydrolases; D004706:Endodeoxyribonucleases; D004720:Endonucleases; D005090:Exodeoxyribonucleases; D005092:Exonucleases; C500807:Tat-D nuclease, S cerevisiae; D008274:Magnesium