Analysis of non-template-directed nucleotide addition and template switching by DNA polymerase.
DNA polymerases use an uninterrupted template strand to direct synthesis of DNA. However, some DNA polymerases can synthesize DNA across two discontinuous templates by binding and juxtaposing them, resulting in synthesis across the junction. Primer/template duplexes with 3' overhangs are especially efficient substrates, suggesting that DNA polymerases use the overhangs as regions of microhomology for template synapsis. The formation of these overhangs may be the result of non-template-directed nucleotide addition by DNA polymerases. To examine the relative magnitude and mechanism of template switching, we studied the in vitro enzyme kinetics of template switching and non-template-directed nucleotide addition by the 3'-5' exonuclease-deficient large fragment of Escherichia coli DNA polymerase I. Non-template-directed nucleotide addition and template switching were compared to that of standard primer extension. We found that non-template-directed nucleotide addition and template switching showed similar rates and were approximately 100-fold slower than normal template-directed DNA synthesis. Furthermore, non-template-directed nucleotide addition showed a 10-fold preference for adding dAMP to the ends of DNA over that of the other three nucleotides. For template switching, kinetic analysis revealed that the two template substrates acted as a random bireactant system with mixed-type inhibition of substrate binding by one substrate over the other. These data are the first to establish the binding kinetics of two discontinuous DNA substrates to a single DNA polymerase. Our results suggest that although the activities are relatively weak, non-template-directed nucleotide addition and template switching allow DNA polymerases to overcome breaks in the template strand in an error-prone manner.
García
Patty B
PB
Department of Biology, Santa Clara University, Santa Clara, California 95053-0268, USA.
Robledo
Nicole L
NL
Islas
Angel L
AL
D004247:DNA / Q000096:biosynthesis; D004256:DNA Polymerase I / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D009711:Nucleotides / Q000378:metabolism*
D009711:Nucleotides; D004247:DNA; D004256:DNA Polymerase I