Dinucleotide repeat expansion catalyzed by bacteriophage T4 DNA polymerasein vitro

DNA replication normally occurs with high fidelity, but certain "slippery" regions of DNA with tracts of mono-, di-, and trinucleotide repeats are fre quently mutation hot spots. We have developed an in vitro assay to study th e mechanism of dinucleotide repeat expansion. The primer-template resembles a base excision repair substrate with a single nucleotide gap centered opp osite a tract of nine CA repeats; nonrepeat sequences flank the dinucleotid e repeats. DNA polymerases are expected to repair the gap, but further exte nsion is possible if the DNA polymerase can displace the downstream oligonu cleotide. We report here that the wild type bacteriophage T4 DNA polymerase carries out gap and strand displacement replication and also catalyzes a d inucleotide expansion reaction. Repeat expansion was not detected for an ex onuclease-deficient T4 DNA polymerase or for Escherichia coil DNA polymeras e I. The dinucleotide repeat expansion reaction catalyzed by wild type T4 D NA polymerase required a downstream oligonucleotide to "stall" replication and 3' --> 5' exonuclease activity to remove the 3'-nonrepeat sequence adja cent to the repeat tract in the template strand. These results suggest that dinucleotide repeat expansion may be stimulated in vivo during DNA repair or during processing of Okazaki fragments.