Functions And Regulation Of Nucleases DNA2and TATDN1in Nucleus And Mitochondrion

DNA2is a nuclease and helicase, which takes part in different pathways during DNA replication and repair in yeast, such as RNA primer removal during okazaki fragment maturation, DSB end resection and chicken foot stabilization. In2008, our lab had found that human DNA2also has conserved nuclease and helicase domain but has no classic NLSs, so our immunoflorescense and fractionation results showed that human DNA2majorly localized in mitochondria but not nucleus. In vitro assay suggested that hDNA2had the potential removing RNA primer during mtDNA replication and promoting poly catalytic activity to enhance the DNA synthesis. Furthermore, hDNA2was also a candidate nuclease to take part in the LP-BER pathway in mitochondria to maintain the mtDNA stability. Consistent with these results, an Italy group collaborating with us has found that myopathy patients carried several conserved amino acid mutations on DNA2, and these mutants had nuclease or helicase defective in biochemical assay, so we predicted that these DNA2mutants may cause mitochondrial disease directly or indirectly.After these discovers, our lab had established the DNA2knockout mouse model. Interestingly, DNA2homozygous knockout mice were lethal at embryo, suggesting that DNA2is critical during mouse development, but the DNA2heterozygous knockout mice were viable. To our surprise, though mouse DNA2also lost all the NLSs, DNA2deficiency lead to telomere replication defects, such as telomere loss, fragile telomeres, sister telomere associations and so on. In vitro assay showed that DNA2can efficiently resovle G4-quardruplex, which is formed by telomere specific repeat sequence TTAGGG, and G4structure was much more stabilized by G4stable molecular in DNA2+/-mice MEF cells. Moreover, DNA2deficiency induced telomere damage and chromosome segregation error, resulting in tetraploidy and aneuploidy. Consequently, DNA2+/-mice developed into cancers containing dysfunctional telomeres.Post-translational modifications always play important roles to regulate protein functions. Phosphorylations in yeast DNA2were reported to recruit DNA2to DSB foci, repairing the damaged DNA. Mammalian DNA2PTMS were little investigated, here we focus on ubiquitination of hDNA2. When DNA suffers damage, DNA repair proteins are triggered and recruited to the damage site to repair the damaged DNA. hDNA2was regulated by TRAF6(ubiquitin E3ligase), which ubiquitinated DNA2by a poly-ubiquitin chain, the ubiquitinated DNA2was much more stable and play an important role in DNA repair.Zebrafish TATDN1(zTATDN1) possesses a novel endonuclease activity, which first makes a nick at the DNA duplex and subsequently converts the nick into a DNA double-strand break in vitro. This biochemical property allows zTATDN1to catalyze decatenation of catenated kinetoplast DNA to produce separated linear DNA in vitro. We further determine that zTATDN1is predominantly expressed in eye cells during embryonic development. Knockdown of TATDN1in zebrafish embryos results in an abnormal cell cycle progression, formation of polyploidy and aberrant chromatin structures. Consequently, the TATDN1-deficient morphants have disordered eye cell layers and significantly smaller eyes compared with the WT control. Altogether, our current studies suggest that zTATDN1plays an important role in chromosome segregation and eye development in zebrafish.