Purification and Characterization of the DNA Polymerase α Associated Exonuclease:  The <i>RTH1</i> Gene Product

Zhu, Fan; Biswas, Esther E.; Biswas, Subhasis B.

doi:10.1021/bi962889v

Cited by 30 publications

(23 citation statements)

References 25 publications

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“…To date, nine different proteins involved in DNA metabolic pathways have been reported to interact with hFEN-1 nuclease. They include PCNA, APE-1, Dna2 helicase, RPA, replication factor C (RF-C), pol ␣, pol ␤, pol ⑀, and type II DNA topisomerase (13)(14)(15)(16)(17)(18). All of these proteins are essentially involved in two different general cellular processes, namely DNA replication and DNA base damage repair, which is consistent with the dual roles of hFEN-1 in DNA replication and repair.…”

supporting

confidence: 54%

Stimulation of Eukaryotic Flap Endonuclease-1 Activities by Proliferating Cell Nuclear Antigen (PCNA) Is Independent of Its in Vitro Interaction via a Consensus PCNA Binding Region

Frank

Qiu

Zheng

et al. 2001

Journal of Biological Chemistry

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Interaction between human flap endonuclease-1 (hFEN-1) and proliferating cell nuclear antigen (PCNA) represents a good model for interactions between multiple functional proteins involved in DNA metabolic pathways. A region of 9 conserved amino acid residues (residues Gln-337 through Lys-345) in the C terminus of human FEN-1 (hFEN-1) was shown to be responsible for the interaction with PCNA. Our current study indicates that 4 amino acid residues in hFEN-1 (Leu-340, Asp-341, Phe-343, and Phe-344) are critical for human PCNA (hPCNA) interaction. A conserved PCNA interaction motif in various proteins from assorted species has been defined as Q 1 X 2 X 3 (L/I) 4 X 5 X 6 F 7 (F/Y) 8 , although our results fail to implicate Q 1 (Gln-337 in hFEN-1) as a crucial residue. Surprisingly, all hFEN-1 mutants, including L340A, D341A, F343A, and F344A, retained hPCNA-mediated stimulation of both exo-and flap endonuclease activities. Furthermore, our in vitro assay showed that hPCNA failed to bind to the scRad27 (yeast homolog of FEN-1) nuclease. However, its nuclease activities were significantly enhanced in the presence of hPCNA. Four additional Saccharomyces cerevisiae scRad27 mutants, including multiple alanine mutants and a deletion mutant of the entire PCNA binding region, were constructed to confirm this result. All of these mutants retained PCNA-driven nuclease activity stimulation. We therefore conclude that stimulation of eukaryotic hFEN-1 nuclease activities by PCNA is independent of its in vitro interaction via the PCNA binding region.DNA replication and repair are critical for maintaining genome stability. These processes are in part dependent on the activities of an emerging family of structure-specific endonucleases. These enzymes, typified by eukaryotic flap endonuclease-1 (FEN-1), 1 are substrate structure-specific and multifunctional (1-3). They possess both flap-specific endonuclease and nick-specific exonuclease (ribonuclease) activities and interact with proliferating cell nuclear antigen (PCNA) as well as other proteins (3-5). This unique substrate-specific nature is based on structural elements that have been observed in the threedimensional structures of prokaryotic FEN-1 homologues (6 -10). The N-terminal region and the region located in between the two conserved nuclease motifs three-dimensionally form an arch, thus creating a hole in the middle of the protein. The dimensions of the hole only allow single-stranded DNA, but not double-stranded DNA, to thread through. The presence of positively charged and bulky residues in the helices of the arch region may directly interact with DNA substrate. Other structural features, such as the H3TH motif, dynamically hold the double-stranded portion of the flap substrate (7). Therefore, hFEN-1 nuclease is able to recognize the ends of free 5Ј single strand DNA and thread it through the hole, resulting in cleavage at the junction between the single-stranded and doublestranded DNA portions of the substrate (11). At the cellular level, hFEN-1 nuclease is able to loca...

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confidence: 54%

Stimulation of Eukaryotic Flap Endonuclease-1 Activities by Proliferating Cell Nuclear Antigen (PCNA) Is Independent of Its in Vitro Interaction via a Consensus PCNA Binding Region

Frank

Qiu

Zheng

et al. 2001

Journal of Biological Chemistry

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“…RTH1 nuclease possesses a RNase H activity that is most likely important in the maturation of Okazaki fragments in the lagging strand of the replication fork, similar to that observed with the 5′ f 3′ exonuclease activity of DNA polymerase I of E. coli (Kornberg & Baker, 1992). Earlier we have shown that RTH1 nuclease can degrade multimeric RNA primers synthesized by yeast pol R-primase complex (Zhu et al, 1997). Therefore, we have examined the effect of RPA on the RNase H activity of RTH1 nuclease.…”

Section: Native Rth1 Endonuclease Prefers Dna Substrates Having a Flamentioning

confidence: 99%

“…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). The native RTH1 5′ f 3′ exonuclease also possesses a structure-specific endonuclease activity on forked DNA structures, in addition to its exonuclease and RNase H activities.…”

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confidence: 92%

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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“…Rad27, a fourth, related protein, is a structure-specific endoand exonuclease homologous to the human FEN-1 enzyme (5,21,31,42,51,55,71). Yeast cells carrying a null allele of the RAD27 gene display a broad array of defects in genome stability, including an elevated spontaneous mutation rate (64), expansion and contraction of micro-and minisatellite sequences (19,26,27,48,56), telomeric repeat fluctuation (40), increased spontaneous recombination (19,32,55,64,67), and increased chromosome breakage (19,67).…”

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confidence: 99%

Novel Function of Rad27 (FEN-1) in Restricting Short-Sequence Recombination

Negritto¹,

Qiu

Ratay³

et al. 2001

Molecular and Cellular Biology

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Saccharomyces cerevisiae mutants lacking the structure-specific nuclease Rad27 display an enhancement in recombination that increases as sequence length decreases, suggesting that Rad27 preferentially restricts recombination between short sequences. Since wild-type alleles of both RAD27 and its human homologue FEN1 complement the elevated short-sequence recombination (SSR) phenotype of a rad27-null mutant, this function may be conserved from yeast to humans. Furthermore, mutant Rad27 and FEN-1 enzymes with partial flap endonuclease activity but without nick-specific exonuclease activity partially complement the SSR phenotype of the rad27-null mutant. This suggests that the endonuclease activity of Rad27 (FEN-1) plays a role in limiting recombination between short sequences in eukaryotic cells.

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

Cited by 30 publications

References 25 publications

Stimulation of Eukaryotic Flap Endonuclease-1 Activities by Proliferating Cell Nuclear Antigen (PCNA) Is Independent of Its in Vitro Interaction via a Consensus PCNA Binding Region

Stimulation of Eukaryotic Flap Endonuclease-1 Activities by Proliferating Cell Nuclear Antigen (PCNA) Is Independent of Its in Vitro Interaction via a Consensus PCNA Binding Region

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

Novel Function of Rad27 (FEN-1) in Restricting Short-Sequence Recombination

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