Human DNA polymerase mu (Pol mu) exhibits an unusual replication slippage ability at AAF lesion.
We analyzed the ability of various cell extracts to extend a radiolabeled primer past an N-2-acetylaminofluorene (AAF) adduct located on a primed single-stranded template. When the 3' end of the primer is located opposite the lesion, partially fractionated human primary fibroblast extracts efficiently catalyzed primer-terminus extension by adding a ladder of about 15 dGMPs, in an apparently non-templated reaction. This activity was not detected in SV40-transformed fibroblasts or in HeLa cell extracts unless purified human DNA polymerase mu (Pol mu) was added. In contrast, purified human Pol mu alone could only add three dGMPs as predicted from the sequence of the template. These results suggest that a cofactor(s) present in cellular extracts modifies Pol mu activity. The production of the dGMP ladder at the primer terminus located opposite the AAF adduct reveals an unusual ability of Pol mu (in conjunction with its cofactor) to perform DNA synthesis from a slipped intermediate containing several unpaired bases.
Duvauchelle
Jean-Baptiste
JB
UPR9003 du CNRS, Cancérogenèse et Mutagenèse Moléculaire et Structurale, ESBS, Boulevard S. Brant, 67400 Strasbourg, France.
Blanco
Luis
L
Fuchs
Robert P P
RP
Cordonnier
Agnes M
AM
D015073:2-Acetylaminofluorene / Q000378:metabolism*; D000906:Antibodies / Q000494:pharmacology; D001483:Base Sequence; D002273:Carcinogens / Q000378:metabolism*; D002457:Cell Extracts / Q000032:analysis; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D018736:DNA Adducts / Q000378:metabolism*; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000276:immunology / Q000378:metabolism*; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008957:Models, Genetic; D019384:Nucleic Acid Synthesis Inhibitors; D012333:RNA, Messenger / Q000096:biosynthesis
D000906:Antibodies; D002273:Carcinogens; D002457:Cell Extracts; D018736:DNA Adducts; D003848:Deoxyguanine Nucleotides; D003854:Deoxyribonucleotides; D019384:Nucleic Acid Synthesis Inhibitors; D012333:RNA, Messenger; C007257:2'-deoxyguanosine 5'-phosphate; D015073:2-Acetylaminofluorene; C407743:DNA polymerase mu; D004259:DNA-Directed DNA Polymerase