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 alpha from the yeast Saccharomyces 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, 3020-3027]. Peptide sequence analysis of the purified 47 kDa exonuclease was carried out, and the peptide sequence was found to be identical to the S. cerevisiae gene YKL510 encoded polypeptide, which is also known as yeast RAD2 homolog 1 or RTH1 nuclease. The native exonuclease also had strong flap endonuclease activity similar to that observed with RTH1 nuclease and homologous yeast (RAD2) and mammalian enzymes. During our studies, we have discovered certain unique features of the mechanism of action of the native RTH1 nuclease. Studies presented here indicated that the exonuclease had specific pause sites during its 5'-3' exonuclease nucleotide excision. These pause sites were easily detected with long (approximately 50 bp) oligonucleotide substrates during exonucleolytic excision by the formation of a discontinuous ladder of excision product. We have further analyzed the mechanism of generation of the pause sites, as they could occur through a number of different pathways. Alignment of the pause sites with the nucleotide sequence of the oligonucleotide substrate indicated that the pause sites were dependent on the nucleotide sequence. Our analysis revealed that RTH1 nuclease pauses predominantly at G:C rich sequences. With poly(dA):oligo(dT)50 as substrate, the exonucleolytic products formed a continuous ladder with no evidence of pausing. The G:C rich DNA sequences are thermodynamically more stable than the A:T rich sequences, which may be in part responsible for pausing of the RTH1 5' --> 3' exonuclease at these sites.
Zhu
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Department of Molecular Biology, University of Medicine & Dentistry of New Jersey, Stratford 08084, USA.
Biswas
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Biswas
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D000595:Amino Acid Sequence; D001483:Base Sequence; D004257:DNA Polymerase II / Q000302:isolation & purification* / Q000378:metabolism; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000302:isolation & purification* / Q000378:metabolism; D005800:Genes, Fungal; D007700:Kinetics; D008969:Molecular Sequence Data; D016914:Ribonuclease H / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017421:Sequence Analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D004257:DNA Polymerase II; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; D016914:Ribonuclease H