Zhu F X, Biswas E E, Biswas S B
Department of Molecular Biology, University of Medicine & Dentistry of New Jersey, Stratford 08084, USA.
Biochemistry. 1997 May 20;36(20):5947-54. doi: 10.1021/bi962889v.
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.
我们在此报告了从酿酒酵母中纯化并对与DNA聚合酶α相关的5'-3'核酸外切酶进行机理表征的研究。此前，我们鉴定出一种5'→3'核酸外切酶活性，它在一个多蛋白复合物中与酵母DNA聚合酶α-引发酶共同纯化[Biswas, E. E., 等人 (1993) 《生物化学》, 32, 3020 - 3027]。对纯化的47 kDa核酸外切酶进行了肽序列分析，发现该肽序列与酿酒酵母基因YKL510编码的多肽相同，该多肽也被称为酵母RAD2同源物1或RTH1核酸酶。天然核酸外切酶还具有很强的瓣状内切核酸酶活性，类似于RTH1核酸酶以及同源的酵母（RAD2）和哺乳动物酶所观察到的活性。在我们的研究过程中，我们发现了天然RTH1核酸酶作用机制的某些独特特征。此处呈现的研究表明，该核酸外切酶在其5'-3'核酸外切酶核苷酸切除过程中有特定的暂停位点。在通过形成不连续的切除产物梯进行核酸外切切除过程中，使用长（约50 bp）寡核苷酸底物时很容易检测到这些暂停位点。我们进一步分析了暂停位点产生的机制，因为它们可能通过多种不同途径出现。将暂停位点与寡核苷酸底物的核苷酸序列进行比对表明，暂停位点取决于核苷酸序列。我们的分析表明，RTH1核酸酶主要在富含G:C的序列处暂停。以聚（dA）:寡聚（dT）50为底物时，核酸外切产物形成连续的梯，没有暂停的迹象。富含G:C的DNA序列在热力学上比富含A:T的序列更稳定，这可能部分解释了RTH1 5'→3'核酸外切酶在这些位点的暂停现象。
