Requirement of ATM-Dependent Phosphorylation of Brca1 in the DNA Damage Response to Double-Strand Breaks
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Supplemental information is available at www.sciencemag.org/feature/data/1044763.shl.
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GST-Brca1 expression vectors were constructed in pGEX2Tkcs or pGEX5X3 by polymerase chain reaction (PCR) with the following primers: pDC78 (Brca1 1 to 500) GAAAGCATATGATGGATTTATCTGCTCTTCGC and GAAAGCTCGAGTTAATTTGTGAGGGGACGCTC; pDC79 (Brca1 452 to 1079) GAAAGCATATGGTAGAGAGTAATATTGAAGAC and GAAAGCTCGAGCTATTTTGGCCCTCTGTTTCT; pDC80 (Brca1 1021 to 1552) GAAAGCATATGAGTACAGTGAGCACAATTAGC and GAAAGCTCGAGCTAGTAAGATGTTTCCGTCAA; pDC81 (Brca1 1501 to 1861) GAAAGCATATGTGCCCATCATTAGATGATAGG and GAAAGCTCGAGTCAGTAGTGGCTGTGGGGGAT; pDC113 (Brca1 1021 to 1211) GAAAGGATCCCAAGTACAGTGAGCACAATTAGCCG and GAAAGGTCGACGGACTCTAATTTCTTGGCCCCTC; pDC114 (Brca1 1211 to 1351) GAAAGGACCAGTCCTCAGAAGAGAACTTATCTAG and GAAAGGTCGACCAAGCCCGTTCCTCTTTCTTCATC; and pDC115 (Brca1 1351 to 1552) GAAAGGATCCGCTTGGAAGAAAATAATCAAGAAGA and GAAAGGTCGACGTAAGATGTTTCCGTCAAATCGTG. In vitro kinase assays were performed essentially as described (9). The ATM expression constructs were a kind gift of M. Kastan.
Single-letter abbreviations for the amino acid residues are as follows: A Ala; C Cys; D Asp; E Glu; F Phe; G Gly; H His; I Ile; K Lys; L Leu; M Met; N Asn; P Pro; Q Gln; R Arg; S Ser; T Thr; V Val; W Trp; and Y Tyr.
Flag-tagged NLS-Brca1 1351 to 1552 was constructed with the univector plasmid fusion system (29). The host vector is based on pCMV2-Flag (Sigma). The SV40 large T antigen NLS was inserted between the Hind III and Not I sites with the following primers: AGCTTCCCAAGAAGAAGAGGAAGGC and GGCCGCCTTCCTCTTCTTCTTGGGA. pUNI15 Brca1 1351 to 1552 was made with the same primers as those for pDC115 described above. The serine to alanine mutations were made by single-stranded mutagenesis with the following primers: S1423A ACAGCATGGGGCCCAGCCTTCTAACAG; and S1524A AAACTACCCAGCTCAAGAGGAACTCATTAAGGTTGTT. All mutations and PCR products were sequenced. Transfections were performed by standard calcium phosphate technique (30).
pBABEpuro HABrca1 was made by digesting pCDNA3βHABrca1 (16) with Sal I and Xho I and inserting it into the Sal I site of pBABEpuro. The serine to alanine mutations were inserted into wild-type Brca1 in pBABEpuro by exchanging a Pfl MI to Apa I Brca1 fragment from the mutated gene into pBABEpuro HABrca wild type. This deletes a portion of Brca1 because an Apa I site had been created by the introduction of the S1423A mutation. This deleted Brca1 segment was reintroduced as an Apa I to Apa I fragment derived from the full-length S1423A/S1524A mutant in pBSKII(−) reconstituting full-length HABrca1 S1423A/S1524A. Retrovirus was made by cotransfection of the pBABEpuro vectors with amphotrophic packaging DNA into 293T cells essentially as described (30). Viral supernatants were used to infect the HCC1937 cells. Two days after infection cells were selected for puromycin resistance with puromycin (1 μg/ml; Sigma).
We thank D. Livingston R. Scully M. Kastan and Y. Shiloh for providing reagents and M. Huang for helpful comments on the manuscript. D.C. is a Fellow of the Jane Coffin Childs Memorial Fund for Medical Research. This work was supported by grants GM44664 and Q1187 (Welch) to S.J.E. and grant IRG199A (American Cancer Society) and a grant from the L.E. Gordy Cancer Research Fund to J.Q. S.J.E is an Investigator with the Howard Hughes Medical Institute.