Redundancy in response to DNA damage: the key to protection of genome integrity.

Progression through the cell cycle is mediated by cyclin-dependent kinases (Cdks) that are activated by their respective cyclins. The cyclin D-Cdk4/6 and, subsequently, cyclin E-Cdk2 phosphorylate retinoblastoma tumor suppressor protein (pRb), leading to a release E2F transcription factors from the complex with pRB. The release turns on transcription of genes coding for proteins required to initiate cell cycle progression and enter the S phase.1 Availability of cyclins, whose content is regulated transcriptionally and post-transcriptionally, is the opening mechanism of modulation of Cdks' activity.2 Subsequent regulation is provided by the two families of cyclin-Cdk inhibitors (CKI), the INK4 and Kip/Cip proteins. The INK4 inhibitors (p15, p16, p19) target cyclin D-Cdk4/6 complexes preventing entry to cell cycle (G0 to G1 transition). The Kip/Cip proteins (p21, p27, p57) inactivate cyclin E-Cdk2, thereby blocking the G1 to S transition. Damage to DNA triggers several signaling pathways defined as the DNA damage response (DDR). Activation of these pathways has four critical goals: (1) halting cell cycle progression to prevent transfer of DNA damage to progeny cells; (2) increasing accessibility of the damage sites to repair machinery; (3) engagement of repair machinery and (4) preparing apoptotic response to eliminate cells whose DNA has not been successfully repaired.3 For the fail-safe execution of these goals, individual steps of DDR are highly redundant and use diversity of mechanism. The capability to phosphorylate the same proteins by ATM, ATR, DNA-PKcs or Chk2 protein kinases is a classic example of such redundancy. Similarly, halting cell cycle progression in response to induction of p53 can be achieved by inactivation of cyclin E-Cdk2 by p21 as well as through Cdc25A and Cdc25C phosphatases that dephosphorylate cyclin E-Cdk2. Numerous redundant pathways, ubiquitin-dependent as well -independent, are involved in regulation of abundance of p21 protein. Ciznadija et al.,4 in a recent Cell Cycle article, address this subject and describe mechanisms modulating turnover of p21 upon induction of p53 in response to DNA damage by ionizing radiation. In elegant experiments utilizing multiparameter flow cytometry, cell sorting, elutriation and gel blot analysis of proteins from the sorted cells, the authors correlated expression of p53, p21, p27, Chk2 and hdm2 vis-a-vis cell cycle phase in the irradiated cells.4 Similar to the case of DNA damage induced by DNA topoisomerase I inhibitor camptothecin, the damage by radiation led to induction of p53 in cells in all phases across the cell cycle.5 However, the accumulation of p21 was limited to cells in G1 and G2 phases. The apparent reduction of p21 seen in S-phase cells was mediated by the proteasome-dependent turnover pathway but independent of SCFSKP2 E3 ubiquitin ligase activity. Using hdm2 siRNA and hdm2 knockout cells, the authors also explored the role of hdm2 in turnover of p21 in the S phase in irradiated cells. Interestingly, similar to the case of ionizing radiation, the induction of DNA damage by UV was shown in another study to lead to proteosomal degradation of p21 also in S-phase cells.6 The degradation was also independent of SCFSKP2 but was mediated by the CRL4CDT2 ligase, whose activity, in this case, required the interaction of p21 with PCNA.6 However, in the S phase of non-irradiated cells, a redundancy between CRL4CDT2 and SCFSKP2 in their ability to ubiquitilate p21 has been observed.6 Other ubiquitin ligases, such as APC/CCDC20, CRKLRR1, DBD1CUL4, target nuclear p21 as well and play a role in its turnover,7 while CLR2LRR1 targets cytoplasmic p21.8 The possible involvement of these ligases in stability of p21 after DNA damage has not been investigated. Regulation of p21 turnover is also mediated by phosphorylation by the nuclear-Dbf2-related (NDR) kinases, the enzymes that are highly conserved from yeast to human.9 Little is known about their possible involvement in p21 turnover after induction of DNA damage, specifically whether their activity contributes to the reduction of p21 expression in S-phase cells. The multiplicity of mechanisms regulating abundance of p21 after induction of DNA damage, their redundancies and evolutionary stability all suggest that, highly controlled and likely associated with multiple signaling pathways related to the cell cycle phase, regulation of p21 turnover is essential for effective DNA repair and maintenance of genome integrity.