In cells, sequence-specific transcription factors must search through an entire genome to find their target sites in promoters. Such sites may be identified by using one-dimensional (linear diffusion) and/or three-dimensional (association/dissociation) mechanisms. We show here that wild-type p53 possesses the ability to linearly diffuse on DNA. p53 lacking its C terminus is incapable of such sliding along DNA, while the isolated C terminus of p53 is even more effective than the full-length protein at one-dimensional linear diffusion. Importantly, neither acetylation-mimicking mutations nor phosphorylation of residues within the C terminus stimulates linear diffusion by p53. Supporting these in vitro observations, we found that C-terminally deleted p53 (p53Delta30) expressed at physiological levels is deficient in binding to and transactivating downstream promoters in vivo. Therefore, our data show that the C terminus is a positive regulator of DNA binding in vivo and in vitro, and indicate that the mechanism may involve linear diffusion.

DNA damage tolerance facilitates the progression of replication forks that have encountered obstacles on the template strands. It involves either translesion DNA synthesis initiated by proliferating cell nuclear antigen monoubiquitination or less well-characterized fork reversal and template switch mechanisms. Herein, we characterize a novel tolerance pathway requiring the tumor suppressor p53, the translesion polymerase ι (POLι), the ubiquitin ligase Rad5-related helicase-like transcription factor (HLTF), and the SWI/SNF catalytic subunit (SNF2) translocase zinc finger ran-binding domain containing 3 (ZRANB3). This novel p53 activity is lost in the exonucleasedeficient but transcriptionally active p53(H115N) mutant. Wild-type p53, but not p53(H115N), associates with POLι in vivo. Strikingly, the concerted action of p53 and POLι decelerates nascent DNA elongation and promotes HLTF/ZRANB3-dependent recombination during unperturbed DNA replication. Particularly after cross-linkerinduced replication stress, p53 and POLι also act together to promote meiotic recombination enzyme 11 (MRE11)-dependent accumulation of (phospho-)replication protein A (RPA)-coated ssDNA. These results implicate a direct role of p53 in the processing of replication forks encountering obstacles on the template strand. Our findings define an unprecedented function of p53 and POLι in the DNA damage response to endogenous or exogenous replication stress.T he tumor suppressor protein p53 has been called the guardianof-the-genome due to its ability to transactivate downstream targets transcriptionally, which prevents S-phase entrance before facilitating DNA repair or eliminating cells with severe DNA damage via apoptosis (1). Interestingly, p53 also encodes an intrinsic 3′-5′ exonuclease activity located within its central DNA-binding domain (2-4). The contribution of the exonuclease proficiency to p53's function has largely remained obscure. Exonucleases are involved in DNA replication, DNA repair, and recombination, increasing the fidelity or efficiency of these processes. The 3′-5′ exonuclease activity of DNA polymerases (POLs) catalyzes the correction of replication errors, thereby preventing genomic instability and cancer (5-7). The potential involvement of p53's exonuclease in DNA repair has been ascribed to transcription-independent functions in nucleotide excision repair and base excision repair, in homologous recombination (HR), and in mitochondrial processes (8-10).Regarding HR, in particular, reports indicate a dual role for p53. On the one hand, it has been reported that p53 down-regulates unscheduled and excessive HR in response to severe genotoxic stress, like formation of DNA double-strand breaks (DSBs) (8-10). This antirecombinogenic effect of p53 has been linked to the blockage of continued strand exchange by interactions with recombinase RAD51, RAD54, and nascent HR intermediates carrying specific mismatches (11, 12). On the other hand, p53 stimulates spontaneous HR during S-phase to overcome replication fork stalling and to pr...

p53 is required for the induction of a G(1) and/or G(2) irreversible arrest after gamma irradiation (IR), whereas blocked DNA replication causes a p53-independent S-phase arrest. We have examined the response to p53 when DNA synthesis is blocked by hydroxyurea (HU) or aphidicolin or when DNA is damaged by gamma IR. Similarly to gamma IR, blocked DNA synthesis induces high levels of phosphorylated nuclear p53. Surprisingly, several (but not all) p53 transcriptional targets that are rapidly induced by gamma IR are weakly or not induced when DNA replication is blocked. Moreover, the p53 response to gamma IR is inhibited by pretreatment of cells with HU or aphidicolin, suggesting that blocked DNA replication prevents p53 from being fully active as a transcription factor. HU-induced stabilization of p53 neither requires functional ATM (ataxia telangiectasia mutated), nor interferes with the gamma IR-dependent activation of the ATM kinase. Thus, stalled replication forks activate kinases that modify and stabilize p53, yet act downstream of ATM to impair p53 transcriptional activity. The ramifications of this novel regulation of p53 are discussed.

Although the contribution of mitochondrial dynamics (a balance in fusion/fission events and changes in mitochondria subcellular distribution) to key biological process has been reported, the contribution of changes in mitochondrial fusion to achieve efficient steroid production has never been explored. The mitochondria are central during steroid synthesis and different enzymes are localized between the mitochondria and the endoplasmic reticulum to produce the final steroid hormone, thus suggesting that mitochondrial fusion might be relevant for this process. In the present study, we showed that the hormonal stimulation triggers mitochondrial fusion into tubular-shaped structures and we demonstrated that mitochondrial fusion does not only correlate-with but also is an essential step of steroid production, being both events depend on PKA activity. We also demonstrated that the hormone-stimulated relocalization of ERK1/2 in the mitochondrion, a critical step during steroidogenesis, depends on mitochondrial fusion. Additionally, we showed that the SHP2 phosphatase, which is required for full steroidogenesis, simultaneously modulates mitochondrial fusion and ERK1/2 localization in the mitochondrion. Strikingly, we found that mitofusin 2 (Mfn2) expression, a central protein for mitochondrial fusion, is upregulated immediately after hormone stimulation. Moreover, Mfn2 knockdown is sufficient to impair steroid biosynthesis. Together, our findings unveil an essential role for mitochondrial fusion during steroidogenesis. These discoveries highlight the importance of organelles’ reorganization in specialized cells, prompting the exploration of the impact that organelle dynamics has on biological processes that include, but are not limited to, steroid synthesis.

Both fission yeast and mammalian cells require the function of the checkpoint kinase CHK1 for G 2 arrest after DNA damage. The tumor suppressor p53, a well-studied stress response factor, has also been shown to play a role in DNA damage G 2 arrest, although in a manner that is probably independent of CHK1. p53, however, can be phosphorylated and regulated by both CHK1 as well as another checkpoint kinase, hCds1 (also called CHK2). It was therefore of interest to determine whether reciprocally, p53 affects either CHK1 or CHK2. We found that induction of p53 either by diverse stress signals or ectopically using a tetracycline-regulated promoter causes a marked reduction in CHK1 protein levels. CHK1 downregulation by p53 occurs as a result of reduced CHK1 RNA accumulation, indicating that repression occurs at the level of transcription. Repression of CHK1 by p53 requires p21, since p21 alone is sufficient for this to occur and cells lacking p21 cannot downregulate CHK1. Interestingly, pRB is also required for CHK1 downregulation, suggesting the possible involvement of E2F-dependent transcription in the regulation of CHK1. Our results identify a new repression target of p53 and suggest that p53 and CHK1 play interdependent and complementary roles in regulating both the arrest and resumption of G 2 after DNA damage.Detection of DNA damage results in the activation of signaling pathways that trigger cell cycle checkpoints (15,25,31,52). Cell cycle arrest prevents DNA replication and mitosis in the presence of unrepaired chromosomal alterations. The p53 tumor suppressor protein and its transcriptional targets play an important role in both G 1 and G 2 checkpoints in mammalian cells (1,13,41). While G 1 arrest relies extensively on the synthesis of p21 which is induced by p53 (11,12,24,69,70), it has been postulated that p53-mediated G 2 arrest involves not only induction of p21 but other p53 targets such as 14-3-3-(17, 32) and GADD45 (38). Arrest in the G 2 phase of the cell cycle in p53 Ϫ/Ϫ cells is the consequence of the activation of the CHK-Cdc25-Cdc2 pathway (51,54,55), although it has been demonstrated that a prolonged G 2 -M arrest requires p53 as well (13).Mitotic cell cycle checkpoints are conserved between yeasts and mammals. After genotoxic stress such as DNA damage or stalled DNA replication, fission yeast CHK1 and Cds1 effector kinases phosphorylate Cdc25 (25,71,74,78), leading to its inactivation as a result of its association with 14-3-3 proteins (54, 78). The activity of yeast CHK1 is controlled by the upstream kinase Rad3, a phosphatidylinositol 3-kinase family member that has homology with mammalian ATM and ATR genes (42,46,72). The mammalian homologues of CHK1 (26, 57) and hCds1 (also called CHK2) (8, 10, 47) have been identified, and their activation has been shown to require ATM and ATR as well. Arrest in the G 2 phase of the cell cycle in mammals has been shown to require CHK proteins (33,45,64). However, despite the existence of a conserved CHK-Cdc25-Cdc2 pathway, there is increasing evidence tha...

p21Cip1/WAF1 is a known inhibitor of the short-gap filling activity of proliferating cell nuclear antigen (PCNA) during DNA repair. In agreement, p21 degradation after UV irradiation promotes PCNA-dependent repair. Recent reports have identified ubiquitination of PCNA as a relevant feature for PCNA-dependent DNA repair. Here, we show that PCNA ubiquitination in human cells is notably augmented after UV irradiation and other genotoxic treatments such as hydroxyurea, aphidicolin and methylmethane sulfonate. Intriguingly, those DNA damaging agents also promoted downregulation of p21. While ubiquitination of PCNA was not affected by deficient nucleotide excision repair (NER) and was observed in both proliferating and arrested cells, stable p21 expression caused a significant reduction in UVinduced ubiquitinated PCNA. Surprisingly, the negative regulation of PCNA ubiquitination by p21 does not depend on the direct interaction with PCNA but requires the cyclin dependent kinase binding domain of p21. Taken together, our data suggest that p21 downregulation plays a role in efficient PCNA ubiquitination after UV irradiation.

Although p21 upregulation is required to block cell-cycle progression following many types of genotoxic insult, UV irradiation triggers p21 proteolysis. The significance of the increased p21 turnover is unclear and might be associated with DNA repair. While the role of p21 in nucleotide excision repair (NER) remains controversial, recent reports have explored its effect on translesion DNA synthesis (TLS), a process that avoids replication blockage during S phase. Herein, we analyze the effect of p21 on different PCNA-driven processes including DNA replication, NER and TLS. Whereas only the CDK-binding domain of p21 is required for cell-cycle arrest in unstressed cells, neither the CDK-binding nor the PCNA-binding domain of p21 is able to block early and late steps of NER. Intriguingly, through its PCNA-binding domain, p21 inhibits the interaction of the TLS polymerase, pol η (pol eta), with PCNA and impairs the assembly of pol η foci after UV. Moreover, this obstruction correlates with accumulation of phosphorylated H2AX and increased apoptosis. By showing that p21 is a negative regulator of PCNA-pol η interaction, our data unveil a link between efficient TLS and UV-induced degradation of p21.

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