Purification and characterization of a yeast DNA polymerase .alpha. complex with associated primase, 5'.fwdarw.3' exonuclease, and DNA-dependent ATPase activities

Biswas, Esther E.; Chen, Pei Hua; Gray, W. J. H.; Li, Y.H.; Ray, Satyajit; Biswas, Subhasis B.

doi:10.1021/bi00063a012

Cited by 9 publications

(13 citation statements)

References 41 publications

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“…Characterization of the 5′ f 3′ Exonuclease Associated with the DNA Polymerase R Complex. Previously we have purified a complex of DNA polymerase R with an associated 5′ f 3′ exonuclease activity (Biswas et al, 1993a). In order to characterize this nuclease activity, we have purified the protein to homogeneity using a multistep purification procedure, as described above.…”

Section: Resultsmentioning

confidence: 99%

“…We reported earlier the presence of a 5′ f 3′ exonuclease activity that copurified with DNA polymerase R-primase complex from the yeast S. cereVisiae (Biswas et al, 1993a). In order to decipher the role(s) of the exonuclease in the chromosomal DNA replication, it was essential to develop a method of purification of this exonuclease activity.…”

Section: Discussionmentioning

confidence: 99%

“…[R-32 P]ATP, [R-32 P]dATP, and [γ-32 P]ATP were obtained from DuPont/NEN (Boston, MA). RPA used in this study was purified as described (Biswas et al 1993a). Terminal deoxynucleotidyl transferase was purchased from United States Biochemical Corp, (Cleveland, OH).…”

Section: Methodsmentioning

confidence: 99%

“…Yeast extract (Fr I) was prepared from 400 g of log phase protease-deficient yeast (strain BJ2168, Yeast Genetic Stock Center, Berkeley, CA) as previously described (Biswas et al, 1993a). The initial purification of DNA polymerase R-primase complex, with associated 5′ f 3′ exonuclease, DNA-dependant ATPase and helicase activities, was carried out as described earlier (Biswas et al, 1993a). The isolation of the 5′ f 3′ exonuclease was then carried out as follows.…”

Section: Methodsmentioning

confidence: 99%

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Purification and Characterization of the DNA Polymerase α Associated Exonuclease: The RTH1 Gene Product

Zhu

Biswas

1997

Biochemistry

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Purification and Characterization of the DNA Polymerase α Associated Exonuclease: The RTH1 Gene Product

Zhu

Biswas

1997

Biochemistry

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“…Earlier we have shown that a 5′ f 3′ exonuclease copurifies with DNA polymerase R-primase (pol R) complex (Biswas et al, 1993a). Purification of this nuclease from S. cereVisiae and determination of its identity as the RTH1 5′ f 3′ exonuclease have been described earlier (Zhu et al, 1997).…”

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confidence: 94%

Stimulation of RTH1 Nuclease of the Yeast Saccharomyces cerevisiae by Replication Protein A

1997

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The RTH1 nuclease is involved in the replication of chromosomal DNA as well as in the repair of DNA damage. Replication protein A (RPA) is also an integral part of the DNA replication and repair processes. We have investigated the roles(s) of RPA in the function of RTH1 nuclease, including its structure specific endonuclease activity. Initial in Vitro studies, which employed a "flap" or a "pseudo Y" substrate containing a short 14 bp duplex region, showed the effect of RPA to be minimal or inhibitory. As RPA inhibition is unwarranted for a protein participating in the DNA replication process, we have further investigated the mechanism of such inhibition. Alternate flap and pseudo Y substrates with a long duplex region (50 bp) were prepared using M13mp19 ssDNA and synthetic oligonucleotides. Yeast RPA stimulated the endonuclease activity of RTH1 endonuclease with these substrates in a dose-dependent manner. Kinetic analysis suggested that yRPA exerted a bipartite effect on the nuclease reaction: (i) the "load time" of RTH1 nuclease onto the DNA substrate decreased from ∼5 to 2 min in the presence of RPA, and (ii) following initiation of the nuclease reaction, the initial rate of the reaction increased 10-fold in the presence of yRPA. Further analysis of the interaction of RPA with various endonuclease substrates indicated that RPA has a weak helix destabilizing effect and could melt small, 14 bp, regions of duplex DNA. RTH1 endonuclease cleaves the DNA strand at the junction of single-and doublestranded DNA; consequently, the observed inhibition with small duplex substrates was likely due to duplex melting. Our studies also demonstrated that RPA stimulated the RNase H activity of RTH1 nuclease significantly. In both instances (RTH1 endonuclease and RNase H), the stimulation may involve a specific interaction of RPA with the RTH1 nuclease rather than a structural positioning of the DNA substrate by RPA.

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Biochemical, cellular and molecular identification of DNA polymerase α in yeast mitochondria

Lasserre¹,

Plissonneau²,

Velours³

et al. 2013

Biochimie

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Purification and characterization of a yeast DNA polymerase .alpha. complex with associated primase, 5'.fwdarw.3' exonuclease, and DNA-dependent ATPase activities

Cited by 9 publications

References 41 publications

Purification and Characterization of the DNA Polymerase α Associated Exonuclease: The RTH1 Gene Product

Purification and Characterization of the DNA Polymerase α Associated Exonuclease: The RTH1 Gene Product

Stimulation of RTH1 Nuclease of the Yeast Saccharomyces cerevisiae by Replication Protein A

Biochemical, cellular and molecular identification of DNA polymerase α in yeast mitochondria

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