The findings confirm and extend these observationsThe findings confirm and extend these observations

The p53 tumor suppressor inhibits HR in a variety of human and murine cell lines {Bertrand, 1997 #23} {Mekeel, 1997 #25} {Gebow, 2000 #44} {Wiese, 2001 #45}. Multiple findings have suggest that p53 may be directly involved in the regulation of HR {Susse, 2000 #28} {Dudenhoffer, 1999 #48} {Sirbu, 2011 #35} {Hampp, 2016 #29}. It was recently established that binding with RPA is critical for the inhibition of spontaneous and replication arrest-induced HR by p53 {Romanova, 2004 #34} {Sirbu, 2011 #35}, and the p53(53.54) mutant with disrupted RPA-binding domain fails to inhibit these HR pathways. In the cells undergoing genotoxic stress, the stability of the p53 complex with RPA is regulated by phosphorylation of p53 at Ser37/46 by ATR/ATM as well as by phosphorylation of RPA2 at Ser4/8 by DNA-PKCS {Serrano, 2013 #36}. While phosphorylation of both proteins leads to dissolution of the p53 and RPA2 complex and upregulation of HR, phosphorylation-deficient mutants stabilize the complex and inhibit HR. The importance of RPA2 phosphorylation for efficient DNA repair by HR in cells undergoing replication arrest was emphasized by other studies {Shi, 2010 #49} {Vassin, 2004 #19} {Anantha, 2007 #14} {Vassin, 2009 #50}. Our findings confirm and extend these observations to show that phosphorylation of RPA2 and therefore, the stability of the p53 complex with RPA is regulated by p53 through transcriptional activation of its downstream target, p21Waf1.  As expected, changes in stability of p53 complex with RPA are accompanied by changes in HR rates. This mechanism of HR regulation by p53 is distinct from the previously reported transactivation-dependent cell cycle re-distribution and transactivation-independent p53/RPA binding, although it regulates the latter.In p53 proficient cells, we observed a delay of RPA2 phosphorylation at S29 as well as Tyr21 and Ser4/8 that correspond to CDK2, ATR/ATM, and DNA-PKcs consensus sites. Being a potent inhibitor of CDKs, an activated p53 probably exerts its effect initially through inhibition of RPA2 phosphorylation at S29, which is followed by a delay in phosphorylation of other sites. This idea is supported by prior observations of inter-dependent phosphorylation of various sites within the RPA2 subunit {Anantha, 2007 #14}. The authors showed that CPT-induced phosphorylation of RPA2 at S23 and S29 by CDK2 is required for the subsequent RPA2 phosphorylation by ATR/ATM and DNA-PKcs at residues Thr21, Ser33 and Ser4/8.  The sequential RPA2 phosphorylation is required for efficient HR DNA repair in response to replication arrest {Vassin, 2004 #19} {Anantha, 2007 #14} {Murphy, 2014 #58}.  A sequential RPA2 phosphorylation during DNA repair may limit the use of recombinant RPA2 mutants “locked” in any phosphorylation state for investigation of this phenomenon. In fact, both phosphorylation-deficient as well as phosphorylation-mimetic RPA2 mutants abolish HR DNA repair when they replace the endogenous protein {Vassin, 2004 #19} {Shi, 2010 #49} {Lee, 2010 #51}.Both A549 and U2OS cells significantly delay S phase progression in response to CPT, pointing at the activation of S phase checkpoint. Moreover, p53-proficient cells proceed through S phase with a similar pace as those deficient in p53 and p21Waf1. Others have shown that p53 status does not affect cell cycle progression in similar settings {Furuta, 2006 #39}. This result is probably expected as p53 is not involved in regulation of the S phase checkpoint.  On the other hand, phosphorylation of Thr21 and Ser4/8 is diminished in the parental cells compare to its p53- and p21Waf1-compromised derivatives. This may reflect the activation status of ATR, ATM, and DNA-PKcs that regulate the S phase checkpoint by stimulating CHK1 and CHK2 signaling. Although a correlation between ATR/ATM and DNA-PKcs-dependent S phase checkpoint activation and RPA2 phosphorylation was observed {Shao, 1999 #52}, these events could be uncoupled {Morgan, 1997 #53} {Olson, 2006 #16}. Thus, our data suggest that the status of RPA2 phosphorylation is not indicative of S phase checkpoint activity, but rather reflects the intensity of DNA repair. In fact, as judged by g-H2AX immunostaining, we identified a faster removal of double-strand breaks from p53- and p21waf1 depleted cells than from intact ones.             In our prior publication, we reported that the conformational and the DNA contact p53 mutants, His175 and His273 {Romanova, 2004 #34} efficiently bound non-phosphorylated RPA in unstressed cells. Despite that, both mutants failed to suppress spontaneous HR, thereby suggesting that RPA binding is necessary but not sufficient for inhibition of HR. Inability of both mutants to bind recombination intermediates {Dudenhoffer, 1999 #48} was suggested to constitute an additional requirement for HR inhibition by p53. Here, both mutants were co-expressed in A549 cells carrying wild-type copies of p53. Inhibition of p21Waf1 activity by both mutants that lead to RPA2 hyperphosphorylation and dissociation of the p53/RPA complex should be responsible for the upregulation of HR in this experiment. The inability of both mutants to bind the recombination intermediates {Dudenhoffer, 1999 #48} probably contributes to the observed effect. The mechanism of HR inhibition by the p53/RPA complex is unknown. It is plausible that RPA recruitment to the ssDNA regions and its progressive phosphorylation would release p53. Phosphorylated forms of RPA acquire an affinity to Rad51 that closely collaborates with RPA at the initial stages of HR when the RPA-coated ssDNA template strand invades the homologous DNA strand and RAD51 searches for homology and strand pairing.  In such settings, RPA brings p53 in close contact with recombination intermediates and Rad51. p53 is known to bind recombination intermediates and Holliday junctions in vitro showing preferences for heteroduplexes that contain nucleotide mismatches. Due to its intrinsic 3′-5′ endonuclease activity, p53 eliminates mispaired nucleotides, which may serve to prevent error-prone exchange processes {Dudenhoffer, 1998 #21} {Janus, 1999 #54} {Susse, 2000 #28}.  Such “proofreading” is accompanied by inhibition of the bacterial analog of Rad51 (reviewed in {Gatz, 2006 #55}). The combined evidence suggests that p53 may collaborate with RPA and Rad51 at the initial stages of recombination processing, probably being directly involved in proofreading and eliminating errors and thus slowing down the recombination process.

            HR is considered to be one of the most accurate DNA repair mechanisms. One would expect that upregulated HR in the cells functionally deficient in p53 would contribute to genome stability. In reality, p53 depletion destabilizes the genome. To address this inconsistency, other p53 effects should be taken into consideration, including regulation of cell cycle arrest that provides additional time for DNA repair, apoptosis, a suggested HR proofreading by p53, and its involvement in nucleotide- and base-excision repairs (reviewed in {Gatz, 2006 #55}). In this context, the p53 deficient cells lacking efficient cell cycle arrest have to rely on upregulated HR to repair double-strand breaks and to survive genotoxic stress. However, in the absence of the other p53-mediated DNA repair, the surviving cells would accumulate multiple genomic rearrangements leading to genomic instability. The outcome of CPT therapy is only a specific manifestation of the general phenomenon. An upregulated HR in the CPT-treated p53/p21Waf1-depleted cells contributes to survival and propagation of the cells carrying various types of DNA damage. This is evident when low concentrations of DNA replication inhibitors are employed. In fact, low concentrations of the CPT analog, etoposide, lead to a senescence-like G2 arrest of p53-positive cells, and to continued proliferation of p53-deficient cells {Marusyk, 2007 #56}. Survival of the latter cells crucially depends on Rad51 and ongoing HR.  The absence of G2 arrest provides these cells with a clonal advantage as compared to the p53 wild-type cells. Our findings of the mechanisms underlying the regulation of HR by the p53/p21Waf1 contribute to an understanding of the drug resistance to the CPT therapy. 

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