Functional Characterization of PARP Dependent Ubls and Dubs Regulating DNA Damage Response

Abstract

DNA damages induced by either endogenous or exogenous factors lead to alteration of DNA structure causing mutation of DNA. Thus, DNA mutations cause a wide range of human disease such as cancers and neuronal disorders. Hence, all organisms have internal surveillance system for monitoring of DNA lesions, thereby control DNA damage response (DDR) for the error-free repair of genetic code. In the DDR, post-translational modifications play the central roles for signal transmission for DNA restoration. Particularly, Poly(ADP-ribose) (PAR)-substrates-ubiquitin axis is a pivotal regulatory network for the recruiting of DNA repair machineries to the DNA lesions that regulates DNA repair processes such as homologous recombination (HR) and non-homologous end joining (NHEJ). Although it has been little known that repair process of double strand breaks (DSBs) can be controlled by ubiquitination, but the direct connection between PAR and ubiquitin signal in DDR remains unclear. Herein, using a systematic screening, we identified the PAR coupled ubiquitin E3 ligase (Ubls) and deubiquitylating enzymes (Dubs) in DDR. We identified 15 Ubls and 6 Dubs linked to PAR signaling pathway and further investigated the functional roles of DIZELs and USP39 on DDR. These proteins were rapidly recruited to DNA lesions via direct interaction with PAR moiety. The DIZELs contain bipartite C2H2-type zinc finger Drought-induced 19 (Di19) motif. By systematic experimental approaches, it has been revealed that C2H2(I), the first motif of the two conserved C2H2 Di-19 region plays an important role for generation of unanchored K11 chains (UnK11Ubcs) but later C2H2(II) motif involves in the direct interaction with PAR moieties. Besides, we also found that UnK11Ubcs synthesized by DIZELs is a crucial factor for recruitment of NPL4/VCP complex and 53BP1 at the DNA lesions. In parallel, we revealed that USP39 also strongly binds to PAR polymer through its N-terminal RG repeated motif and regulates HR or NHEJ repair process, in a splicing-dependent or -independent manner, respectively. Furthermore, we found that a spliceosome independent USP39 shows distinct role for the recruitment of XRCC4/LIG4 complex to regulate NHEJ repair process. Taken together, we have identified that PAR-substrates-ubiquitin axis is a master regulatory network for the regulation of Ubls or Dubs activity, thereby allows monitoring and maintaining of genomic stability.