Weak strand displacement activity enables human DNA polymerase beta to expand CAG/CTG triplet repeats at strand breaks.
Using synthetic DNA constructs in vitro, we find that human DNA polymerase beta effectively catalyzes CAG/CTG triplet repeat expansions by slippage initiated at nicks or 1-base gaps within short (14 triplet) repeat tracts in DNA duplexes under physiological conditions. In the same constructs, Escherichia coli DNA polymerase I Klenow Fragment exo(-) is much less effective in expanding repeats, because its much stronger strand displacement activity inhibits slippage by enabling rapid extension through two downstream repeats into flanking non-repeat sequence. Polymerase beta expansions of CAG/CTG repeats, observed over a 32-min period at rates of approximately 1 triplet added per min, reveal significant effects of break type (nick versus gap), strand composition (CTG versus CAG), and dNTP substrate concentration, on repeat expansions at strand breaks. At physiological substrate concentrations (1-10 microm of each dNTP), polymerase beta expands triplet repeats with the help of weak strand displacement limited to the two downstream triplet repeats in our constructs. Such weak strand displacement activity in DNA repair at strand breaks may enable short tracts of repeats to be converted into longer, increasingly mutable ones associated with neurological diseases.
Department of Biological Sciences, Hedco Molecular Biology Laboratories, University of Southern California, Los Angeles 90089-1340, USA.
Michael J
MJ
Hartenstine
Myron F
MF
Goodman
John
J
Petruska
D002384:Catalysis; D004249:DNA Damage; D019951:DNA Polymerase beta / Q000378:metabolism*; D004260:DNA Repair; D006801:Humans; D013942:Thymine Nucleotides / Q000378:metabolism; D019680:Trinucleotide Repeat Expansion
D013942:Thymine Nucleotides; D019951:DNA Polymerase beta; C024157:thymidine 5'-triphosphate