PURIFICATION AND CHARACTERIZATION OF THE DNA-POLYMERASE-ALPHA ASSOCIATED EXONUCLEASE - THE RTH1 GENE-PRODUCT

We report here the purification and mechanistic characterization of a 5'-3' exonuclease associated with DNA polymerase ex from the yeast Sac charomyces cerevisiae. Earlier, we identified a 5' --> 3' exonuclease activity that copurified with yeast DNA polymerase alpha-primase in a multiprotein complex [Biswas, E, E., et al. (1993) Biochemistry, 32, 3 020-3027]. Peptide sequence analysis of the purified 47 kDa exonucleas e was carried out, and the peptide sequence was found to be identical to the S. cerevisiae gene YKL510 encoded polypeptide, which is also kn own as yeast RAD2 homolog 1 or RTH1 nuclease. The native exonuclease a lso had strong flap endonuclease activity similar to that observed wit h RTH1 nuclease and homologous yeast (RAD2) and mammalian enzymes, Dur ing our studies, we have discovered certain unique features of the mec hanism of action of the native RTH1 nuclease, Studies presented here i ndicated that the exonuclease had specific pause sites during its 5'-3 ' exonuclease nucleotide excision. These pause sites were easily detec ted with long (similar to 50 bp) oligonucleotide substrates during exo nucleolytic excision by the formation of a discontinuous ladder of exc ision product. We have further analyzed the mechanism of generation of the pause sites, as they could occur through a number of different pa thways. Alignment of the pause sites with the nucleotide sequence of t he oligonucleotide substrate indicated that the pause sites were depen dent on the nucleotide sequence. Our analysis revealed that RTH1 nucle ase pauses predominantly at G:C rich sequences, With poly(dA):oligo(dT )(50);as substrate, the exonucleolytic products formed a continuous la dder with no evidence of pausing. The G:C rich DNA sequences are therm odynamically more stable than the A:T rich sequences, which may be in part responsible for pausing of the RTH1 5' --> 3' exonuclease at thes e sites.