p53 and recombination intermediates: role of tetramerization at DNA junctions in complex formation and exonucleolytic degradation.
Heteroduplex joints represent intermediates of Rad51-dependent recombination processes, which are recognized by p53 with extremely high affinities, in a manner independent of the DNA sequence content. To determine the structural elements required for complex formation, we monitored DNA-binding by protection against restriction endonuclease cleavage. We show that wild-type (wt) p53 interacts with heteroduplex joints in the proximity of the flexible junction. Association of p53 within this junction region was also observed with preformed Rad51-heteroduplex complexes, whereas SSB counteracted p53 binding. At a distance of 31 bp from the junction p53 established very few contacts with the heteroduplex, despite the presence of an A-G mismatch. Consistently, p53-dependent exonucleolytic degradation decreased when we raised the distance between the junction and the heteroduplex terminus by 27 bp. Different from the cancer-related mutant p53(273H), which did not recognize the junction, tetramerization defective p53-1262 was protection competent but displayed reduced complex stability in gel shifts. Moreover, p53-1262 performed exonucleolytic activities towards ssDNA like wtp53, but reduced degradation of heteroduplex joints. These results suggest that during recombination wild-type p53, as a tetramer, stably binds to strand transfer regions, enabling the protein to exonucleolytically correct heteroduplex intermediates early after strand invasion.
Janz
Christine
C
Universitätsfrauenklinik und Poliklinik, Prittwitzstrasse 43, D-89075 Ulm, Germany.
Süsse
Silke
S
Wiesmüller
Lisa
L
D000818:Animals; D002460:Cell Line; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000037:antagonists & inhibitors; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D051135:Rad51 Recombinase; D011995:Recombination, Genetic; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009692:Nucleic Acid Heteroduplexes; D016159:Tumor Suppressor Protein p53; D004247:DNA; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D004262:DNA Restriction Enzymes