While p53's role as a transcription factor that controls apoptosis and cell cycle progression is firmly established, a myriad of studies over the past >15 years has ascribed a multitude of additional biochemical and cellular functions to p53 
. A transactivation-independent role of p53 in the downregulation of HR has been reproducibly described by several laboratories, including our own 
. Because careful control of HR activities is important for the response to stalled or collapsed replication forks, elucidating the role of p53 in HR is critical for a better understanding of tumor initiation and progression.
We show here for the first time that p53 downregulates HR in response to replicative stress in a manner that is independent of its role as a transcription factor (, , ). Our data are consistent with the idea that p53's role in HR is dependent on interactions with RPA and ATR kinase, thus implicating p53 in the ATR replication checkpoint (, ). Overall, the anti-recombinogenic functions of the replication checkpoint remain to be fully established 
. In fission yeast, the Chk1 homologue inhibits Mus81 and Rad60 function, thereby preventing undesired recombination 
. In higher eukaryotes, ATR phosphorylates BLM, a known anti-recombinogenic factor 
. On the other hand, ATR has been shown to promote HR 
. Consistent with these data, our findings imply that both ATR and ATM promote RAD51 foci formation in response to replicative stress in a p53-independent fashion (). Thus, there may exist a positive and negative (via p53) regulation of HR by ATR.
With regard to potential limitations of our work, an inherent limitation of foci studies is that they cannot directly measure protein activities at replication forks (, , ). However, foci endpoints are widely used in the literature to determine molecular mechanisms and genetic determinants of HR 
. Second, a similar limitation applies to our plasmid system (), which may not be an accurate measure of physiological HR events that are under p53 control. Third, while all of our and other data suggest that the human p53QS mutant and mouse p53-A135V (or human homolog) are functionally equivalent in terms of suppressing HR in a transactivation-independent manner (for example, Figure S2
, we cannot exclude the possibility that unknown differences may exist. Lastly, we also caution that results obtained with one cell line, such as H1299 lung cancer cells in this study, may not be readily generalized to other cell lines.
What are the molecular mechanisms by which S15 phosphorylation of p53 could suppress HR? In a previously published model, sequestration of RPA from ssDNA will inhibit the subsequent loading of RAD51, and thus is one means by which p53 suppresses HR 
. The p53 N-terminus competes with ssDNA for the OB-fold domain of RPA1's N-terminus 
. Thus, we speculate that mechanisms may exist by which N-terminal phosphorylation of p53 promotes the binding to RPA1, thereby affecting the ssDNA-binding affinity of the DNA binding domains of RPA. For example, altered ssDNA-RPA binding could lead to unscheduled release of RPA from ssDNA impairing with proper RAD51 loading, or p53 may trap RPA on ssDNA and delay RAD51 loading. There are strong interdependencies between the N-terminal p53 phosphorylation sites 
. In H1299 cells, mutating S15 leads to reduced S37 phosphorylation after irradiation 
. Interestingly, Lowry et al. recently found evidence of a collapsed region in the intrinsically unstructured p53 domain with a loop structure centered around residues 34–36 
. These authors suggested that S37 phosphorylation may lead to an open conformation of this domain and thereby promote binding to RPA1. Thus, mutation of S15 would impair HR indirectly through an inhibitory effect on adjacent S37 phosphorylation.
The notion that p53 may suppress DSB repair has come from a series of studies looking at the effect of p53 on site-directed DSB in chromosomally integrated plasmid substrates 
. We found that the magnitude of the suppressive p53 effect is correlated with the length of sequence homology present (, S2
). We postulate that the pDT219/pΔ2 system () is representative of sister chromatid repair because of the extent of available sequence homology is in the kilobase range. While we acknowledge that a comparison between different recombination systems has caveats, a dependence of p53's suppressive effect on homology length is in excellent agreement with a prior study by Wiesmüller et al. 
. These authors, who used a panel of chromosomal EGFP-based substrates, demonstrated that the downregulation of gene conversion events by p53 was particularly pronounced when the length of shared homology was reduced to 168–233 bp. It is possible that p53 creates a threshold between short and long homologies, which may aid in preventing error-prone repair and detrimental rearrangements by misalignment of repetitive DNA. Such a model would be consistent with the observation that cellular p53 status has no direct effect on gene targeting and sister chromatid exchanges, which typically are mediated by long homologies in the order of kilobases 
. This model also predicts that p53 will not negatively affect the repair of chromatid DSB caused by ionizing radiation or other agents, which is in line with cell survival data 
. Our data also suggest that HR proficiency measured with an I-SceI based plasmid system such as pDR-GFP (Figure S2
) is not always a good surrogate marker for HR-dependent repair of exogenous DNA damage. Further, recent data suggest that HR repair of chromosomal I-SceI-induced DSB is cell cycle dependent and subject to transcriptional regulation by p53 (Rieckmann et al., unpublished 2011). Thus, it is possible that HR activities in response to replication stress or frank DSB are differentially regulated by wild-type and mutant p53 variants. We can also not rule out that the regulatory effects may vary between cell lines.
Lastly, p53 did not compromise the RAD51 foci response and cell survival following exposure to the crosslinking agent MMC (). To the contrary, there was even a slight increase in cell survival upon expression of transactivation-deficient p53 mutants (, S6
). The underlying mechanism remains to be determined but may relate to a possible stabilization of multi-protein complexes at the replication fork by p53 (LMM, HW, unpublished data). The observed increase in MMC resistance is consistent with other reports showing that, in the absence of apoptosis, the presence of p53 is associated with cisplatin resistance 
. In contrast, in cell systems or assays susceptible to apoptosis, resistance to DNA damaging agents is typically caused by loss of wild-type p53 
. Overall, the role of p53 in determining cell survival in response to DNA damages is clearly complex and a reflection of p53's multiple functions in apoptosis, cell-cycle control, and DNA recombination. The study of these questions in defined cell systems is a promising avenue of investigation with potential clinical relevance for the treatment of malignant tumors most of which have lost p53 function. In addition, the biological significance of p53's function in HR regulation, especially with regard to its role in tumor suppression, remains to be established.