A new assay to quantify in vivo repair of G:T mispairs by base excision repair.
The double mismatch reversion (DMR) assay quantifies the repair of G:T mispairs exclusively by base excision repair in vivo. Synthetic oligonucleotides containing two G:T mispairs on opposite strands were placed into the suppressor tRNA gene supF in the shuttle plasmid pDMR. Placement of two mispairs on opposite strands of supF creates a one to one correspondence between the number of correct repair events prior to replication in which G:T mispairs are converted to G:C base pairs and the number of post-replication progeny plasmids with functional supF. Replication of unrepaired or incorrectly repaired mispairs cannot produce progeny plasmids containing functional supF. Indeed, direct transformation of Escherichia coli strain MBL50, which reports the functional status of supF, with pDMR constructs containing two G:T or G:G mispairs yielded <0.5% wild-type supF-containing colonies. In contrast, passage of G:T mispair-containing pDMR constructs through human 5637 bladder carcinoma cells for 48h prior to plasmid recovery and transformation of the reporter E. coli strain MBL50 produced 47% wild-type supF-containing colonies. This finding was indicative of repair prior to the onset of replication in 5637 cells. However, passage of G:G mispair-containing pDMR constructs through 5637 cells yielded <0.5% wild-type supF-containing colonies. Moreover, no difference was observed in the rate of G:T mispair repair by HCT 116 colorectal carcinoma cells deficient in long-patch mismatch repair and a long-patch mismatch repair proficient HCT 116 subline. These data demonstrate that repair measured by the DMR assay is exclusively attributable to short-patch pathways. The DMR assay proved useful in the analysis of the effect of the base 5' to a mispaired G on the rate of G:T base excision repair by 5637 cells, indicating the sequence preference CpG approximately 5mCpG>TpG>GpG approximately ApG, and in the comparison of G:T base excision repair rates between cell lines.
Waters
S B
SB
Department of Cancer Biology, Wake Forest University School of Medicine, Medical Center Blvd, Winston-Salem, NC 27157, USA.
Akman
S A
SA
D000230:Adenocarcinoma / Q000235:genetics* / Q000473:pathology; D020137:Base Pair Mismatch; D002295:Carcinoma, Transitional Cell / Q000235:genetics / Q000473:pathology; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation; D004260:DNA Repair; D004261:DNA Replication; D004926:Escherichia coli / Q000235:genetics; D016153:Genes, Suppressor; D016158:Genes, p53; D005821:Genetic Techniques; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000235:genetics / Q000473:pathology
D009838:Oligodeoxyribonucleotides; D012329:RNA, Bacterial; C096275:supF tRNA; D012343:RNA, Transfer