DNA synthesis on discontinuous templates by human DNA polymerases: implications for non-homologous DNA recombination

• Published in: Nucleic acids research 1998-08, Vol.26 (16), p.3729-3738
• Main Authors: Islas, Ángel L., Fairley, Cecilia F., Morgan, William F.
• Format: Article
• Language: English
• Subjects: Animals; Antibodies - pharmacology; Antigens, Nuclear; Base Sequence; CHO Cells; Cricetinae; DNA - biosynthesis; DNA - chemistry; DNA - genetics; DNA Helicases; DNA Polymerase I - antagonists & inhibitors; DNA Polymerase I - immunology; DNA Polymerase I - metabolism; DNA-Activated Protein Kinase; DNA-Binding Proteins - metabolism; DNA-Directed DNA Polymerase - metabolism; Enzyme Inhibitors - pharmacology; Escherichia coli; HeLa Cells; Humans; In Vitro Techniques; Ku Autoantigen; Molecular Sequence Data; Neutralization Tests; Nuclear Proteins - metabolism; Nucleic Acid Synthesis Inhibitors; Protein-Serine-Threonine Kinases - metabolism; Recombination, Genetic; Restriction Mapping; Substrate Specificity; Thermus aquaticus
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/26.16.3729

DNA polymerases catalyze the synthesis of DNA using a continuous uninterrupted template strand. However, it has been shown that a 3′→5′ exonuclease-deficient form of the Klenow fragment of Escherichia coli DNA polymerase I as well as DNA polymerase of Thermus aquaticus can synthesize DNA across two unlinked DNA templates. In this study, we used an oligonucleotide-based assay to show that discontinuous DNA synthesis was present in HeLa cell extracts. DNA synthesis inhibitor studies as well as fractionation of the extracts revealed that most of the discontinuous DNA synthesis was attributable to DNA polymerase α. Additionally, discontinuous DNA synthesis could be eliminated by incubation with an antibody that specifically neutralized DNA polymerase α activity. To test the relative efficiency of each nuclear DNA polymerase for discontinuous synthesis, equal amounts (as measured by DNA polymerase activity) of DNA polymerases α, β, δ (± PCNA) and ε (±PCNA) were used in the discontinuous DNA synthesis assay. DNA polymerase α showed the most discontinuous DNA synthesis activity, although small but detectable levels were seen for DNA polymerases δ (+PCNA) and δ (− PCNA). Klenow fragment and DNA polymerase β showed no discontinuous DNA synthesis, although at much higher amounts of each enzyme, discontinuous synthesis was seen for both. Discontinuous DNA synthesis by DNA polymerase α was seen with substrates containing 3 and 4 bp single-strand stretches of complementarity; however, little synthesis was seen with blunt substrates or with 1 bp stretches. The products formed from these experiments are structurally similar to that seen in vivo for non-homologous end joining in eukaryotic cells. These data suggest that DNA polymerase α may be able to rejoin double-strand breaks in vivo during replication.