Fidelity of a Human Cell DNA Replication Complex

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 85; no. 19; pp. 7064 - 7068
• Main Authors: Roberts, John D., Kunkel, Thomas A.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 01.10.1988; National Acad Sciences
• Subjects: 550201 - Biochemistry- Tracer Techniques; ANTIGENS; Antigens, Polyomavirus Transforming - metabolism; BACTERIA; BASIC BIOLOGICAL SCIENCES; BETA DECAY RADIOISOTOPES; BETA-MINUS DECAY RADIOISOTOPES; Cell Line; Codon; CODONS; DAYS LIVING RADIOISOTOPES; DNA; DNA Polymerase II - metabolism; DNA POLYMERASES; DNA Primase; DNA REPLICATION; ELECTROPHORESIS; ENZYME ACTIVITY; ENZYMES; Error rates; ESCHERICHIA COLI; Genetic mutation; HELA CELLS; Humans; ISOTOPES; LIGHT NUCLEI; MICROORGANISMS; MOLECULAR BIOLOGY; Molecules; MUTAGENESIS; MUTATION FREQUENCY; NUCLEI; NUCLEIC ACID REPLICATION; NUCLEOTIDYLTRANSFERASES; ODD-ODD NUCLEI; Opal; PARASITES; PHOSPHORUS 32; PHOSPHORUS ISOTOPES; PHOSPHORUS-GROUP TRANSFERASES; POLYMERASES; RADIOISOTOPES; RNA Nucleotidyltransferases - metabolism; SIMIAN VIRUS; TRANSFERASES; Viral tumor antigens; VIRUSES
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.85.19.7064

We have measured the fidelity of bidirectional, semiconservative DNA synthesis by a human DNA replication complex in vitro. Replication was performed by extracts of HeLa cells in the presence of simian virus 40 (SV40) large tumor antigen by using a double-stranded phage M13mp2 DNA template containing the SV40 origin of replication and either of two different target sequences for scoring mutations in the lacZα -complementation gene, which encodes the α region (specifying the amino-terminal portion) of β -galactosidase. Replicative synthesis was substantially more accurate than synthesis by the human DNA polymerase α --DNA primase complex purified from HeLa cell extracts by immunoaffinity chromatography, suggesting that additional factors or activities in the extract may increase fidelity during bidirectional replication. However, by using a sensitive opal codon reversion assay, single-base substitution errors were readily detected in the replication products at frequencies significantly higher than estimated spontaneous mutation rates in vivo. These data suggest that additional fidelity factors may be present during chromosomal replication in vivo and/or that the fidelity of replication alone does not account for the low spontaneous mutation rates in eukaryotes.