A DNA POLYMERASE-ALPHA PAUSE SITE IS A HOT-SPOT FOR NUCLEOTIDE MISINSERTION

In this study we examined whether the arrest of DNA polymerase-alpha (pol-alpha)-catalyzed DNA synthesis at template pause sites entails terminal nucleotide misincorporation. An approach was developed to identify the 3'-terminal nucleotide in nascent DNA chains that accumulate at pause sites. A radioactive 5'-end-labeled primer was annealed to a bacteriophage M13mp2 single-stranded DNA template and elongated by pol-alpha. Individual DNA chains that were accumulated at pause sites were resolved by sequencing gel electrophoresis, isolated, and purified. These DNA chains were elongated by pol-alpha by using four annealed synthetic DNA templates, each of which contained a different nucleotide at the position opposite the 3' terminus of the arrested chain. Owing to the high preference of pol-alpha for matched-over-mismatched primer termini, only those templates that contain a nucleotide that is complementary to the 3' terminus of the isolated pause-site chain are copied. Electrophoresis of product DNA showed the extent of copying of each template and thus identified the 3'-terminal nucleotide of the pause-site chains. We found that product DNA chains terminate with a noncomplementary 3'-terminal nucleotide opposite pause sites within the sequence 3'-d(AAAA)-5' at positions 6272-6269 of the M13mp2 genome. pol-alpha catalyzed misincorporation of dG or dA into the 3' terminus of nascent chains opposite two of the M13mp2 template dA residues. A similar analysis of a different pause site did not reveal significant misincorporation opposite template dC. These results suggest that some but not all sites at which pol-alpha pauses may constitute loci of mutagenesis.