Research
Cellular Functions of Poly(ADP-ribosyl)ation Reactions
Introduction
All organisms depend on the integrity of their genome. The preservation of the correct genetic information is crucial, since all cellular functions require the expression of proteins encoded in the DNA. However, DNA is exposed to a number of damaging agents of both endogenous and environmental origin, particulary under certain pathophysiological circumstances. In addition to several preventive mechanisms, nature has developed an impressive array of pathways to repair damage to the genome. The immediate recognition of damage and the initiation of DNA repair are important so that repair can be completed before further damage occurs. It is obvious that it may be advantageous to postpone even vital processes such as replication or transcription until DNA repair has been completed. According to a large body of evidence the nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP-1) participates in the regulation of both DNA repair and transcription. In response to the appearance of DNA lesions, generated either directly by genotoxic agents or indirectly following enzymatic incision of a DNA-base lesion the enzymatic activity of PARP-1 is induced. PARP-1 catalyzes the transfer of ADP-ribose moieties to protein acceptors and synthesis of poly(ADP-ribose) using NAD + as a substrate (Fig. 1). In vivo, PARP-1 is associated with components of the base excision repair (BER) pathway and plays an important role in regulating BER. Poly(ADP-ribose) synthesized by PARP-1 after the occurrence of DNA lesions serves as an energy source for the final and rate-limiting step of BER, DNA-ligation [1].
ATP for the DNA Ligation Step Can Be Generated from Poly(ADP-ribose) and DNA Synthesis
It was shown [1] that poly(ADP-ribose) may serve as an energy source for the final and rate-limiting step of BER, DNA-ligation. This conclusion was drawn from experiments in which the fate of 32P-poly(ADP-ribose) or [a 32P]-NAD + added to HeLa nuclear extracts was systematically followed. ATP was synthesized from poly(ADP-ribose) in a pathway that strictly depended on nick-induced DNA-synthesis. NAD + was used for the synthesis of poly(ADP-ribose) which, in turn, was converted to ATP by pyrophosphorylytic cleavage utilizing the pyrophosphate generated from dNTPs during DNA synthesis. The adenylyl moiety was then preferentially used to adenylate DNA ligase III from which it then was transferred to the 5'-phosphoryl end of the nicked DNA. Finally, ligation to the 3'-OH end resulted in the release of AMP.
State 1: The initial cleavage steps in the BER pathway result in the activation of Pol ß and PARP-1 activities owing to their high affinity to nicked DNA. Gap filling catalyzed by Pol ß yields pyrophosphate from dNTPs. Simultaneously, PARP-1 synthesizes poly(ADP-ribose) ([Rib-P-P-Ado] n ) from NAD+ under release of nicotinamide (Nam). Poly(ADP-ribose) is then degraded and the adenylyl moiety used to form ATP in conjunction with the pyrophosphate (P-P) generated by Pol ß. State 2: Lig III consumes the ATP to adenylate itself under release of pyrophosphate. This might enhance the binding of the ligase to the DNA nick. Besides its interaction with Lig III, XRCC1 now also associates with PARP-1 and Pol ß, thereby repressing their enzymatic activities [2, 3]. A DNase cleaves the overhanging DNA-flap. State 3: The adenylyl group is transferred from Lig III onto the 5'-phosphoryl group of the DNA-nick. Nicked DNA is then ligated under release of AMP. After DNA-repair is completed the catalytic activities of the BER enzymes are down-regulated.
A Cellular Survival Switch: Poly(ADP-ribosyl)ation Silences Transcription and Stimulates DNA Repair [4]
All living cells are exposed to genotoxic challenges. Several pathways have evolved to protect the genome. A member of a multiprotein complex in DNA base excision repair (BER) is the enzyme poly(ADP-ribose) polymerase (PARP-1). Under conditions of severe DNA damage PARP-1 is activated and utilizes NAD + as substrate, catalyzing the synthesis of poly(ADP-ribose). This nuclear post-translational modification has been considered to function in cellular surveillance of genotoxic stress. Recently, we demonstrated that activated PARP-1 promotes BER by generation of ATP from NAD+ via poly(ADP-ribosyl)ation. This generated ATP can be specifically used for the final ligation step of the DNA repair process [1]. In addition, PARP-1 is known to influence gene expression. This regulation includes silencing of transcription by modification of transcription factors resulting in the inability of these proteins to bind to DNA [2, 3].
State 1: PARP-1 detects and binds with high affinity to DNA lesions such as single strand breaks. The binding causes activation of the catalytic activity. State 2: NAD+ is consumed for the poly(ADP-ribosyl)ation of PARP-1 itself and specific transcription factors (TF). Modification of the transcription factors disables these proteins to bind to their recognition site. Thereby, the formation of transcription initiation complexes is prevented [2]. Automodification of PARP-1 causes the dissociation of the protein from the DNA permitting access of repair enzymes. State 3: DNA repair is conducted. The enzymes are depicted which perform the final steps of base excision repair. Pol ß fills the gap and finally Lig III completes repair by ligating the adjacent 3'- and 5'-ends. The ATP for the ligation is generated from ADP-ribose units and the pyrophosphate liberated by Pol ß [1]. State 4: Poly(ADP-ribose) remaining in modified proteins such as PARP-1 and transcription factors is catabolized. As a result the transcription factors regain affinity to their DNA binding sites and mRNA synthesis by RNA polymerase II (Pol II) may start.
References:
[1] Oei, S.L. and Ziegler, M. (2000) J. Biol. Chem. 275 , 23234-23239.
[2] Oei, S.L., Griesenbeck, J., Ziegler, M., and Schweiger, M.(1998) Biochemistry 37, 1465-1469.
[3] Oei., S.L., Griesenbeck, J., Schweiger, M. and Ziegler, M. (1998) J. Biol. Chem. 273 , 31644-31647.
[4] Ziegler, M. and Oei, S.L. (2001) BioEssays, 23, 543-548.
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