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Dissecting the role of p53 phosphorylation in homologous recombination provides new clues for gain-of-function mutants.

ABSTRACT: Regulation of homologous recombination (HR) represents the best-characterized DNA repair function of p53. The role of p53 phosphorylation in DNA repair is largely unknown. Here, we show that wild-type p53 repressed repair of DNA double-strand breaks (DSBs) by HR in a manner partially requiring the ATM/ATR phosphorylation site, serine 15. Cdk-mediated phosphorylation of serine 315 was dispensable for this anti-recombinogenic effect. However, without targeted cleavage of the HR substrate, serine 315 phosphorylation was necessary for the activation of topoisomerase I-dependent HR by p53. Moreover, overexpression of cyclin A1, which mimics the situation in tumors, inappropriately stimulated DSB-induced HR in the presence of oncogenic p53 mutants (not Wtp53). This effect required cyclin A1/cdk-mediated phosphorylation for stable complex formation with topoisomerase I. We conclude that p53 mutants have lost the balance between activation and repression of HR, which results in a net increase of potentially mutagenic DNA rearrangements. Our data provide new insight into the mechanism underlying gain-of-function of mutant p53 in genomic instability.

SUBMITTER: Restle A

PROVIDER: S-EPMC2532731 | biostudies-other | 2008 Sep

REPOSITORIES: biostudies-other

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Dissecting the role of p53 phosphorylation in homologous recombination provides new clues for gain-of-function mutants.

Restle Anja A Färber Martin M Baumann Cindy C Böhringer Michael M Scheidtmann Karl Heinz KH Müller-Tidow Carsten C Wiesmüller Lisa L

Nucleic acids research 20080812 16

Regulation of homologous recombination (HR) represents the best-characterized DNA repair function of p53. The role of p53 phosphorylation in DNA repair is largely unknown. Here, we show that wild-type p53 repressed repair of DNA double-strand breaks (DSBs) by HR in a manner partially requiring the ATM/ATR phosphorylation site, serine 15. Cdk-mediated phosphorylation of serine 315 was dispensable for this anti-recombinogenic effect. However, without targeted cleavage of the HR substrate, serine 3 ...[more]

PMID: 18697815

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Project description:The majority of TP53 missense mutations identified in cancer patients are in the DNA-binding domain and are characterized as either structural or contact mutations. These missense mutations exhibit inhibitory effects on wild-type p53 activity. More importantly, these mutations also demonstrate gain-of-function (GOF) activities characterized by increased metastasis, poor prognosis, and drug resistance. To better understand the activities by which TP53 mutations, identified in Li-Fraumeni syndrome, contribute to tumorigenesis, we generated mice harboring a novel germline Trp53R245W allele (contact mutation) and compared them with existing models with Trp53R172H (structural mutation) and Trp53R270H (contact mutation) alleles. Thymocytes from heterozygous mice showed that all three hotspot mutations exhibited similar inhibitory effects on wild-type p53 transcription in vivo, and tumors from these mice had similar levels of loss of heterozygosity. However, the overall survival of Trp53R245W/+ and Trp53R270H/+ mice, but not Trp53R172H/+ mice, was significantly shorter than that of Trp53+/- mice, providing strong evidence for p53-mutant-specific GOF contributions to tumor development. Furthermore, Trp53R245W/+ and Trp53R270H/+ mice had more osteosarcoma metastases than Trp53R172H/+ mice, suggesting that these two contact mutants have stronger GOF in driving osteosarcoma metastasis. Transcriptomic analyses using RNA sequencing data from Trp53R172H/+, Trp53R245W/+, and Trp53R270H/+ primary osteosarcomas in comparison with Trp53+/- indicated that GOF of the three mutants was mediated by distinct pathways. Thus, both the inhibitory effect of mutant over wild-type p53 and GOF activities of mutant p53 contributed to tumorigenesis in vivo. Targeting p53 mutant-specific pathways may be important for therapeutic outcomes in osteosarcoma.Significancep53 hotspot mutants inhibit wild-type p53 similarly but differ in their GOF activities, with stronger tumor-promoting activity in contact mutants and distinct protein partners of each mutant driving tumorigenesis and metastasis.

Project description:The Saccharomyces cerevisae RAD3 gene is homolog of human XPD, an essential gene encoding a DNA helicase of the TFIIH complex involved in both nucleotide excision repair (NER) and transcription. Mutant alleles of RAD3 have been identified (rad3-101 and rad3-102) that have partial defects in DNA repair associated with a strong hyper-recombination (hyper-Rec) phenotype. Previous studies showed that the hyper-Rec phenotype associated with rad3-101 and rad3-102 can be explained as a consequence of persistent single-stranded DNA gaps that are converted to recombinogenic double-strand breaks (DSBs) by replication. We have further characterized these events using a system in which the reciprocal products of mitotic recombination between homologs are recovered as red and white sectored colonies. Both rad3-101 and rad3-102 elevate the frequency of sectored colonies about 100-fold. Subsequent mapping of these events shows that three-quarters of crossovers between homologs induced in hyper-Rec rad3 mutants reflect DSBs formed in at the same positions in both sister chromatids (double sister-chromatid breaks, DSCBs). The remainder reflects DSBs formed in single chromatids (single chromatid breaks, SCBs). The ratio of DSCBs to SCBs is similar to that observed for spontaneous recombination events in wild-type cells. In addition to examining crossovers on chromosome V, we mapped 216 unselected genomic alterations throughout the genome including crossovers, gene conversions, deletions, and duplications. We found a significant association between the location of these recombination events and regions with elevated gamma-H2AX. In addition, there was a hotspot for deletions and duplications at the IMA2 and HXT11 genes near the left end of chromosome XV. A comparison of these data with our previous analysis of spontaneous mitotic recombination events suggests that a sub-set of spontaneous events in wild-type cells may be initiated by incomplete NER reactions, and that DSCBs, which cannot be repaired by sister-chromatid recombination, are a major source of mitotic recombination between homologous chromosomes.

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Dissecting the role of p53 phosphorylation in homologous recombination provides new clues for gain-of-function mutants.

Publications

Dissecting the role of p53 phosphorylation in homologous recombination provides new clues for gain-of-function mutants.

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