FACS analysis of the portion of cells with G1, S or G2 DNA content or of apoptotic cells failed to detect significant differences between samples without and with expression of Wtp53, p53(273H) or p53(248W), arguing against HR activation being indirectly caused by the classical tumor suppressor activities (Supplementary Physique 1B). Our data provide new insight into the mechanism underlying gain-of-function of mutant p53 in genomic instability. INTRODUCTION Because of the central role of p53 as a gatekeeper and a caretaker, the protein must be subject to complex control mechanisms that orchestrate the multiple functions of p53 in transcription, cell-cycle control, apoptosis induction and DNA repair (1,2). Posttranslational modification of p53 by protein phosphorylation has been the most extensively studied potential functional switch mechanism, as it occurs at multiple serine and threonine residues in response to genotoxic stress (3,4). Oxaliplatin (Eloxatin) Modification of p53 on serine 15 by ATM and ATR was demonstrated to trigger the cascade of damage-induced phosphorylation and acetylation events that have been implicated in protein stabilization and enhancement of transcriptional transactivation (3,4). However, observations made with knock-in mouse models (5,6) indicated a role for serine 18 Oxaliplatin (Eloxatin) in apoptosis, but not in Mdm2-governed protein stability. Moreover, in several studies, no evidence was found for an essential role of the N-terminal casein kinase 1 (CK1) and ATM/ATR phosphorylation sites in damage-induced transcriptional transactivation (7C9). In addition, when DNA replication was Oxaliplatin (Eloxatin) blocked, p53 became phosphorylated on serine 15, but this was not accompanied by a rise in important target gene products such as p21 (10C12). This suggested that after replication fork stalling, p53 Oxaliplatin (Eloxatin) phosphorylated on serine 15 (p53pSer15) may serve additional functions unrelated to transcriptional transactivation. In support of this hypothesis, colocalization studies indicated that p53pSer15 forms a component of RAD51-specific repair assemblies (11C13). Over the last few years, a large body of evidence has emerged indicating that p53 is usually directly involved in DNA repair, particularly in homologous double-strand break (DSB) repair. First, p53 recognizes three-stranded heteroduplex and four-way Holliday junctions and DNA lesions including mismatches, gaps or DNA ends. The core domain name is required for junction DNA-binding and also harbors an exonuclease activity, the extreme C-terminus stimulates these activities upon mismatch acknowledgement (15,2). Second, p53 Oxaliplatin (Eloxatin) actually and functionally interacts with crucial enzymes and surveillance factors of homologous recombination (HR), namely with RAD51, RAD54, the MRE11 complex, BRCA1, BRCA2 and BLM, and counteracts strand exchange catalyzed by RAD51. Third, using different cell-based test systems, several groups concurrently found that Wtp53 represses inter- and intra-molecular HR, when brought on by DSBs or replication blocking brokers. In contrast, hotspot mutants failed to downregulate these HR activities. The identification of separation-of-function mutations, which experienced lost p53’s transcriptional transactivation and cell-cycle regulatory capacity, but retained HR inhibition, and vice versa, provided further evidence for p53’s direct role in HR control (15,2). A recent report explains transcriptional repression of by direct binding of Wtp53 to a response element within the promoter region (16). This mechanism can only partially explain the role of p53 in HR, because mutations within the p53 conversation site of the RAD51 protein abrogate HR repression by p53 (13). Moreover, p53(138V), which is usually defective in sequence-specific DNA binding, retains the HR-downregulatory effect (17). The biological meaning of this, at first sight, paradoxical activity directed against a fairly safe DNA repair pathway was unveiled by systematic substrate variance, which indicated a fidelity control mechanism directed against DNA exchange processes between divergent sequences (in 15). Unexpectedly, Wtp53 was more recently found to stimulate recombination in the absence of targeted substrate cleavage in a manner depending on topoisomerase I (topo I) (18,19). Spontaneous recombination events are coupled to the normal DNA metabolism in proliferating cells such as during the bypass of low level, endogenous lesions at replication forks, which are insufficient to activate stress signalling. Upon exposure to ionizing radiation and generation of highly recombinogenic DNA MYH10 lesions such as DSBs, the serines 6, 15 and 315 symbolize the most prominently phosphorylated p53 residues (3). On the other hand, Subramanian and Griffith (20) exhibited that acknowledgement of Holliday junction DNA by p53 is particularly sensitive to posttranslational phosphorylation at serine 392 as compared to serines 6 or 15. To define the role of phosphorylation in p53-dependent regulation of recombinative repair, we applied an EGFP-based recombination assay in combination with cells expressing the.