The mutational specificity of DNA polymerase-beta during in vitro DNA synthesis. Production of frameshift, base substitution, and deletion mutations.

摘要: The frequency and specificity of mutations produced in vitro by eucaryotic DNA polymerase-beta have been determined a forward mutation assay using 250-base target sequence M13mp2 DNA. Homogeneous polymerase-beta, isolated from four different sources, produces at 4-6%/single round gap-filling synthesis. analyses 460 independent mutants resulting this error-prone synthesis demonstrate wide variety mutational events. Frameshift base substitutions are made approximately equal together comprise about 90% all mutations. Two "hot spots" for frameshift substitution were observed. characteristics the these sites suggest that certain errors result dislocation template bases rather than direct mispair formation polymerase-beta. When considering entire sequence, single-base occur primarily runs identical bases, usually pyrimidines. loss single occurs 20-80 times more frequently additions much two or bases. Base many throughout target, representing spectrum formations. Averaged over large number phenotypically detectable sites, error is greater one mistake every 5000 polymerized. Large deletion also observed, 10-fold background, indicating purified polymerases alone capable producing such deletions. These data discussed relation to physical kinetic properties enzymes with respect proposed role polymerase vivo.