p53 Modulation of Homologous Recombination

Linke SP, Sengupta S, Khabie N, Jeffries BA, Buchhop S, Miska S, Henning W, Pedeux R, Wang XW, Hofseth LJ, Yang Q, Garfield SH, Sturzbecher HW, and Harris CC. p53 interacts with hRAD51 and hRAD54, and directly modulates homologous recombination. Cancer Res 63: 2596–605, 2003.

functional homologous recombination (HR) pathway is essential for faithful genomic replication and cell survival, as unrepaired spontaneous or induced double strand breaks (DSBs) tend to be recombinogenic and/or lethal. However, an overactive HR pathway also could be problematic. For example, inappropriate recombination could lead to losses of heterozygosity, translocations, deletions, or duplications. These processes are all commonly observed in human cancers and tumor cell lines, and elevated levels of RAD51 have been documented in some. This suggests that a balance must be struck in the HR pathway between allowing variability and maintaining genetic stability (Bertrand P et al. Trends Genet 20: 235–243, 2004).

When p53 tumor suppressor function is compromised, cells exhibit increased rates of spontaneous and induced HR. p53 transcriptional activation is involved in its well-characterized mediation of cell cycle arrest and apoptosis. However, there is evidence for a p53 transcription-independent function in apoptosis, and p53-dependent inhibition of recombination also appears to be independent of transcription. In fact, p53 is found in complexes with other proteins directly involved in HR processes, including BLM (Bloom syndrome protein), BRCA1, BRCA2, RAD51, RAD52, RAD54, and WRN (Werner syndrome protein). Our study characterizes p53 interactions with the central HR factors, RAD51 and RAD54.

When cells are challenged with certain DNA damaging agents, probing them for specific repair or checkpoint proteins results in a focal nuclear staining pattern. These foci likely represent sites of damage where the proteins have accumulated. RAD51 accumulates in nuclear foci in cells treated with DSB-inducing agents, such as ionizing radiation or neocarzinostatin, or with agents that stall replication fork progression, such as hydroxyurea. Using an antibody specific for an activated form of p53 phosphorylated at serine-15 (p53pSer15), we found a high percentage of colocalization with RAD51, suggesting that activated p53 accumulated at sites of damage and potential HR repair. The nuclear foci likely represent persistent or slowly repaired breaks, which are prone to inappropriate recombination or misrepair. Thus, p53 accumulation at these sites may be crucial to maintain genetic stability.

We showed that RAD51 coimmunoprecipitated with p53 at endogenous levels in normal human cells to verify that it was directly in the repair complex. Using a mixture of RAD54 antibodies, we also demonstrated that nuclear foci of endogenous RAD54 colocalized with RAD51 and p53pSer15 in normal cells. RAD54 coimmunoprecipitated with both RAD51 and p53 under these conditions. In addition, we demonstrated that the p53 C-terminus binds to RAD54 in vitro. Thus, p53 may be capable of binding directly to both RAD51 and RAD54.

During HR, RAD51 polymerizes on an exposed single strand of DNA from one duplex with the assistance of the chromatin remodeling factor RAD54. The resultant nucleoprotein filament then invades the other duplex. Previous work indicated that p53 could bind to the RAD51 homo-oligomerization domain. When we overexpressed RAD51, we observed complex nuclear networks of high-order RAD51 filaments. However, when p53 was coexpressed with RAD51, formation of these filaments was greatly inhibited. Interestingly, the tumor-derived p53/273H mutant, which inefficiently binds RAD51, did not significantly inhibit polymerization. In addition, polymerization of a p53-binding mutant of RAD51 was not inhibited by p53. These data establish in living cells that p53 can modulate RAD51 polymerization through direct binding.

Finally, we conducted a functional assay looking at the effect of p53 and RAD51 expression on recombination between plasmids, which we first validated with dominant-negative mutants of p53, RAD51, and RAD54. As expected, wild-type RAD51 elevated HR levels. Interestingly, a transcriptionally inactive p53 mutant reduced this back to the basal level. However, the p53-binding mutant of RAD51 was not inhibited by p53. Cumulatively, the data indicate that p53 inhibits the polymerization of RAD51, thereby inhibiting HR, in a transcription-independent manner.

Figure 1. Mechanistic model for the restoration of stalled DNA replication forks. A regressed replication fork is restored by either reverse branch migration, mediated by BLM (Bloom syndrome protein) helicase in a non-recombinogenic pathway (indicated by a thick arrow, pathway A), or a recombinogenic pathway that involves endonucleolytic cleavage by one or more resolvases (e.g., Mus81), followed by RAD51/RAD54-mediated homologous recombination (indicated by a thin arrow, pathway B). p53 functions as a “molecular governor” of homologous recombination. The simplified model shows a single DNA lesion at a replication fork that could represent a carcinogen-DNA adduct, a UV photoproduct, or a base damaged by a free radical.

Other studies from our lab indicate that p53 is transported to sites of potential HR by BLM protein. When replication forks stall, fork regression can occur (Figure 1), and BLM can directly reverse this process in the absence of HR to restore the fork. The presence of p53 may assist by inhibiting both RAD51-mediated fork regression and nucleoprotein filament formation, as well as through an interaction with RAD54. Similar inhibition mechanisms may exist for other modes of HR.

There are some limitations of this study due to the artificiality of the RAD51 polymerization and extrachromosomal HR assay. In addition, controversy remains about whether overactive HR contributes to cancer and whether transcription-independent p53 modulation of HR activity is one of p53’s tumor-suppressive functions. Future studies should shed more light on these issues.

Steven P. Linke, PhD
Principal Investigator

Curtis C. Harris, MD
Chief, Laboratory of Human
Carcinogenesis
NCI-Bethesda, Bldg. 37/Rm. 3068
Tel: 301-496-2048
Fax: 301-496-0497
Curtis_Harris@nih.gov

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