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Volume 17, Number 22, Issue of November 15, 1997 pp. 8711-8720

DNA Replication and Postreplication Mismatch Repair in Cell-Free Extracts from Cultured Human Neuroblastoma and Fibroblast Cells

Received July 14, 1997; revised Aug. 25, 1997; accepted Aug. 29, 1997.

Pascale David¹, Edna Efrati¹, Georges Tocco¹, Sharon Wald Krauss², and Myron F. Goodman¹

¹ Department of Biological Sciences, Hedco Molecular Biology Laboratories, University of Southern California, Los Angeles, California 90089-1340, and ² Department of Biophysics and Biomolecular Structure, University of California, Lawrence Berkeley National Laboratory, Berkeley, California 94720

DNA synthesis and postreplication mismatch repair were measured in vitro using cell-free extracts from cultured human SY5Y neuroblastoma and WI38 fibroblast cells in different growth states. All extracts, including differentiated SY5Y and quiescent WI38 fibroblasts, catalyzed SV40 origin-dependent DNA synthesis, totally dependent on SV40 T-antigen. Thus, although differentiated neuroblastoma and quiescent fibroblasts cells were essentially nondividing, their extracts were competent for DNA replication using DNA polymerases , , and possibly , with proliferating cell nuclear antigen. Nonreplicative DNA synthesis and lesion bypass by either - or -polymerases were detected independently in extracts using primed or gapped single-stranded DNA templates. Long-patch postreplication mismatch repair was measured for the first time in neuroblastoma cell-free extracts. Extracts from subconfluent and high-density SY5Y cells catalyzed postreplication mismatch repair with efficiencies comparable to those of HeLa cell extracts. No significant differences were observed in repair between SY5Y differentiated and undifferentiated cell extracts. Mismatch repair efficiencies were threefold lower in extracts from subconfluent WI38 cells, and repair in WI38 quiescent cells was fourfold less than in subconfluent cells, suggesting that mismatch repair may be regulated. The spectrum of mismatch repair in SY5Y extracts closely resembled the mismatch removal specificities of HeLa extracts: T · G and G · G mismatches were repaired most efficiently; C · A, A · A, A · G and a five-base loop were repaired with intermediate efficiency; repair of G · A, C · C, and T · T mismatches was extremely inefficient.

Key words: DNA replication; mismatch repair; abasic lesion bypass; SY5Y neuroblastoma; WI38 fibroblasts; human cell-free extracts; differentiation; Pol ; Pol

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