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Robert A Bambara - One of the best experts on this subject based on the ideXlab platform.
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Msh2-Msh3 Interferes with Okazaki Fragment Processing to Promote Trinucleotide Repeat Expansions
Cell reports, 2012Co-Authors: Athena Kantartzis, Gregory M. Williams, Lata Balakrishnan, Rick L. Roberts, Jennifer A. Surtees, Robert A BambaraAbstract:Summary Trinucleotide repeat (TNR) expansions are the underlying cause of more than 40 neurodegenerative and neuromuscular diseases, including myotonic dystrophy and Huntington's disease. Although genetic evidence points to errors in DNA replication and/or repair as the cause of these diseases, clear molecular mechanisms have not been described. Here, we focused on the role of the mismatch repair complex Msh2-Msh3 in promoting TNR expansions. We demonstrate that Msh2-Msh3 promotes CTG and CAG repeat expansions in vivo in Saccharomyces cerevisiae. Furthermore, we provide biochemical evidence that Msh2-Msh3 directly interferes with normal Okazaki Fragment Processing by flap endonuclease1 (Rad27) and DNA ligase I (Cdc9) in the presence of TNR sequences, thereby producing small, incremental expansion events. We believe that this is the first mechanistic evidence showing the interplay of replication and repair proteins in the expansion of sequences during lagging-strand DNA replication.
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An Alternative Pathway for Okazaki Fragment Processing RESOLUTION OF FOLD-BACK FLAPS BY Pif1 HELICASE
Journal of Biological Chemistry, 2010Co-Authors: Jason E. Pike, Peter M J Burgers, Judith L. Campbell, Ryan A. Henry, Robert A BambaraAbstract:Two pathways have been proposed for eukaryotic Okazaki Fragment RNA primer removal. Results presented here provide evidence for an alternative pathway. Primer extension by DNA polymerase δ (pol δ) displaces the downstream Fragment into an RNA-initiated flap. Most flaps are cleaved by flap endonuclease 1 (FEN1) while short, and the remaining nicks joined in the first pathway. A small fraction escapes immediate FEN1 cleavage and is further lengthened by Pif1 helicase. Long flaps are bound by replication protein A (RPA), which inhibits FEN1. In the second pathway, Dna2 nuclease cleaves an RPA-bound flap and displaces RPA, leaving a short flap for FEN1. Pif1 flap lengthening creates a requirement for Dna2. This relationship should not have evolved unless Pif1 had an important role in Fragment Processing. In this study, biochemical reconstitution experiments were used to gain insight into this role. Pif1 did not promote synthesis through GC-rich sequences, which impede strand displacement. Pif1 was also unable to open fold-back flaps that are immune to cleavage by either FEN1 or Dna2 and cannot be bound by RPA. However, Pif1 working with pol δ readily unwound a full-length Okazaki Fragment initiated by a fold-back flap. Additionally, a fold-back in the template slowed pol δ synthesis, so that the Fragment could be removed before ligation to the lagging strand. These results suggest an alternative pathway in which Pif1 removes Okazaki Fragments initiated by fold-back flaps in vivo.
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Components of the Secondary Pathway Stimulate the Primary Pathway of Eukaryotic Okazaki Fragment Processing
Journal of Biological Chemistry, 2010Co-Authors: Ryan A. Henry, Judith L. Campbell, Lata Balakrishnan, Stefanie Tan Ying-lin, Robert A BambaraAbstract:Reconstitution of eukaryotic Okazaki Fragment Processing implicates both one- and two-nuclease pathways for Processing flap intermediates. In most cases, FEN1 (flap endonuclease 1) is able to efficiently cleave short flaps as they form. However, flaps escaping cleavage bind replication protein A (RPA) inhibiting FEN1. The flaps must then be cleaved by Dna2 nuclease/helicase before FEN1 can act. Pif1 helicase aids creation of long flaps. The pathways were considered connected only in that the products of Dna2 cleavage are substrates for FEN1. However, results presented here show that Dna2, Pif1, and RPA, the unique proteins of the two-nuclease pathway from Saccharomyces cerevisiae, all stimulate FEN1 acting in the one-nuclease pathway. Stimulation is observed on RNA flaps representing the initial displacement and on short DNA flaps, subsequently displaced. Neither the RNA nor the short DNA flaps can bind the two-nuclease pathway proteins. Instead, direct interactions between FEN1 and the two-nuclease pathway proteins have been detected. These results suggest that the proteins are either part of a complex or interact successively with FEN1 because the level of stimulation would be similar either way. Proteins bound to FEN1 could be tethered to the flap base by the interaction of FEN1 with PCNA, potentially improving their availability when flaps become long. These findings also support a model in which cleavage by FEN1 alone is the preferred pathway, with the first opportunity to complete cleavage, and is stimulated by components of the backup pathway.
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acetylation of dna2 endonuclease helicase and flap endonuclease 1 by p300 promotes dna stability by creating long flap intermediates
Journal of Biological Chemistry, 2010Co-Authors: Lata Balakrishnan, Piotr Polaczek, Judith L. Campbell, Jason A. Stewart, Robert A BambaraAbstract:Flap endonuclease 1 (FEN1) and Dna2 endonuclease/helicase (Dna2) sequentially coordinate their nuclease activities for efficient resolution of flap structures that are created during the maturation of Okazaki Fragments and repair of DNA damage. Acetylation of FEN1 by p300 inhibits its endonuclease activity, impairing flap cleavage, a seemingly undesirable effect. We now show that p300 also acetylates Dna2, stimulating its 5′–3′ endonuclease, the 5′–3′ helicase, and DNA-dependent ATPase activities. Furthermore, acetylated Dna2 binds its DNA substrates with higher affinity. Differential regulation of the activities of the two endonucleases by p300 indicates a mechanism in which the acetylase promotes formation of longer flaps in the cell at the same time as ensuring correct Processing. Intentional formation of longer flaps mediated by p300 in an active chromatin environment would increase the resynthesis patch size, providing increased opportunity for incorrect nucleotide removal during DNA replication and damaged nucleotide removal during DNA repair. For example, altering the ratio between short and long flap Okazaki Fragment Processing would be a mechanism for better correction of the error-prone synthesis catalyzed by DNA polymerase α.
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pif1 helicase lengthens some okazaki Fragment flaps necessitating dna2 nuclease helicase action in the two nuclease Processing pathway
Journal of Biological Chemistry, 2009Co-Authors: Jason E. Pike, Peter M J Burgers, Judith L. Campbell, Robert A BambaraAbstract:We have developed a system to reconstitute all of the proposed steps of Okazaki Fragment Processing using purified yeast proteins and model substrates. DNA polymerase δ was shown to extend an upstream Fragment to displace a downstream Fragment into a flap. In most cases, the flap was removed by flap endonuclease 1 (FEN1), in a reaction required to remove initiator RNA in vivo. The nick left after flap removal could be sealed by DNA ligase I to complete Fragment joining. An alternative pathway involving FEN1 and the nuclease/helicase Dna2 has been proposed for flaps that become long enough to bind replication protein A (RPA). RPA binding can inhibit FEN1, but Dna2 can shorten RPA-bound flaps so that RPA dissociates. Recent reconstitution results indicated that Pif1 helicase, a known component of Fragment Processing, accelerated flap displacement, allowing the inhibitory action of RPA. In results presented here, Pif1 promoted DNA polymerase δ to displace strands that achieve a length to bind RPA, but also to be Dna2 substrates. Significantly, RPA binding to long flaps inhibited the formation of the final ligation products in the reconstituted system without Dna2. However, Dna2 reversed that inhibition to restore efficient ligation. These results suggest that the two-nuclease pathway is employed in cells to process long flap intermediates promoted by Pif1.
Judith L. Campbell - One of the best experts on this subject based on the ideXlab platform.
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Okazaki Fragment Processing-Independent Role for Human Dna2 Enzyme During DNA Replication
Journal of Biological Chemistry, 2012Co-Authors: Julien P. Duxin, Judith L. Campbell, Hayley R. Moore, Julia M. Sidorova, Kenneth K. Karanja, Yuchi Honaker, Benjamin Dao, Helen Piwnica-worms, Raymond J. Monnat, Sheila A. StewartAbstract:Dna2 is an essential helicase/nuclease that is postulated to cleave long DNA flaps that escape FEN1 activity during Okazaki Fragment (OF) maturation in yeast. We previously demonstrated that the human Dna2 orthologue (hDna2) localizes to the nucleus and contributes to genomic stability. Here we investigated the role hDna2 plays in DNA replication. We show that Dna2 associates with the replisome protein And-1 in a cell cycle-dependent manner. Depletion of hDna2 resulted in S/G2 phase-specific DNA damage as evidenced by increased γ-H2AX, replication protein A foci, and Chk1 kinase phosphorylation, a readout for activation of the ATR-mediated S phase checkpoint. In addition, we observed reduced origin firing in hDna2-depleted cells consistent with Chk1 activation. We next examined the impact of hDna2 on OF maturation and replication fork progression in human cells. As expected, FEN1 depletion led to a significant reduction in OF maturation. Strikingly, the reduction in OF maturation had no impact on replication fork progression, indicating that fork movement is not tightly coupled to lagging strand maturation. Analysis of hDna2-depleted cells failed to reveal a defect in OF maturation or replication fork progression. Prior work in yeast demonstrated that ectopic expression of FEN1 rescues Dna2 defects. In contrast, we found that FEN1 expression in hDna2-depleted cells failed to rescue genomic instability. These findings suggest that the genomic instability observed in hDna2-depleted cells does not arise from defective OF maturation and that hDna2 plays a role in DNA replication that is distinct from FEN1 and OF maturation.
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An Alternative Pathway for Okazaki Fragment Processing RESOLUTION OF FOLD-BACK FLAPS BY Pif1 HELICASE
Journal of Biological Chemistry, 2010Co-Authors: Jason E. Pike, Peter M J Burgers, Judith L. Campbell, Ryan A. Henry, Robert A BambaraAbstract:Two pathways have been proposed for eukaryotic Okazaki Fragment RNA primer removal. Results presented here provide evidence for an alternative pathway. Primer extension by DNA polymerase δ (pol δ) displaces the downstream Fragment into an RNA-initiated flap. Most flaps are cleaved by flap endonuclease 1 (FEN1) while short, and the remaining nicks joined in the first pathway. A small fraction escapes immediate FEN1 cleavage and is further lengthened by Pif1 helicase. Long flaps are bound by replication protein A (RPA), which inhibits FEN1. In the second pathway, Dna2 nuclease cleaves an RPA-bound flap and displaces RPA, leaving a short flap for FEN1. Pif1 flap lengthening creates a requirement for Dna2. This relationship should not have evolved unless Pif1 had an important role in Fragment Processing. In this study, biochemical reconstitution experiments were used to gain insight into this role. Pif1 did not promote synthesis through GC-rich sequences, which impede strand displacement. Pif1 was also unable to open fold-back flaps that are immune to cleavage by either FEN1 or Dna2 and cannot be bound by RPA. However, Pif1 working with pol δ readily unwound a full-length Okazaki Fragment initiated by a fold-back flap. Additionally, a fold-back in the template slowed pol δ synthesis, so that the Fragment could be removed before ligation to the lagging strand. These results suggest an alternative pathway in which Pif1 removes Okazaki Fragments initiated by fold-back flaps in vivo.
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Components of the Secondary Pathway Stimulate the Primary Pathway of Eukaryotic Okazaki Fragment Processing
Journal of Biological Chemistry, 2010Co-Authors: Ryan A. Henry, Judith L. Campbell, Lata Balakrishnan, Stefanie Tan Ying-lin, Robert A BambaraAbstract:Reconstitution of eukaryotic Okazaki Fragment Processing implicates both one- and two-nuclease pathways for Processing flap intermediates. In most cases, FEN1 (flap endonuclease 1) is able to efficiently cleave short flaps as they form. However, flaps escaping cleavage bind replication protein A (RPA) inhibiting FEN1. The flaps must then be cleaved by Dna2 nuclease/helicase before FEN1 can act. Pif1 helicase aids creation of long flaps. The pathways were considered connected only in that the products of Dna2 cleavage are substrates for FEN1. However, results presented here show that Dna2, Pif1, and RPA, the unique proteins of the two-nuclease pathway from Saccharomyces cerevisiae, all stimulate FEN1 acting in the one-nuclease pathway. Stimulation is observed on RNA flaps representing the initial displacement and on short DNA flaps, subsequently displaced. Neither the RNA nor the short DNA flaps can bind the two-nuclease pathway proteins. Instead, direct interactions between FEN1 and the two-nuclease pathway proteins have been detected. These results suggest that the proteins are either part of a complex or interact successively with FEN1 because the level of stimulation would be similar either way. Proteins bound to FEN1 could be tethered to the flap base by the interaction of FEN1 with PCNA, potentially improving their availability when flaps become long. These findings also support a model in which cleavage by FEN1 alone is the preferred pathway, with the first opportunity to complete cleavage, and is stimulated by components of the backup pathway.
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acetylation of dna2 endonuclease helicase and flap endonuclease 1 by p300 promotes dna stability by creating long flap intermediates
Journal of Biological Chemistry, 2010Co-Authors: Lata Balakrishnan, Piotr Polaczek, Judith L. Campbell, Jason A. Stewart, Robert A BambaraAbstract:Flap endonuclease 1 (FEN1) and Dna2 endonuclease/helicase (Dna2) sequentially coordinate their nuclease activities for efficient resolution of flap structures that are created during the maturation of Okazaki Fragments and repair of DNA damage. Acetylation of FEN1 by p300 inhibits its endonuclease activity, impairing flap cleavage, a seemingly undesirable effect. We now show that p300 also acetylates Dna2, stimulating its 5′–3′ endonuclease, the 5′–3′ helicase, and DNA-dependent ATPase activities. Furthermore, acetylated Dna2 binds its DNA substrates with higher affinity. Differential regulation of the activities of the two endonucleases by p300 indicates a mechanism in which the acetylase promotes formation of longer flaps in the cell at the same time as ensuring correct Processing. Intentional formation of longer flaps mediated by p300 in an active chromatin environment would increase the resynthesis patch size, providing increased opportunity for incorrect nucleotide removal during DNA replication and damaged nucleotide removal during DNA repair. For example, altering the ratio between short and long flap Okazaki Fragment Processing would be a mechanism for better correction of the error-prone synthesis catalyzed by DNA polymerase α.
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pif1 helicase lengthens some okazaki Fragment flaps necessitating dna2 nuclease helicase action in the two nuclease Processing pathway
Journal of Biological Chemistry, 2009Co-Authors: Jason E. Pike, Peter M J Burgers, Judith L. Campbell, Robert A BambaraAbstract:We have developed a system to reconstitute all of the proposed steps of Okazaki Fragment Processing using purified yeast proteins and model substrates. DNA polymerase δ was shown to extend an upstream Fragment to displace a downstream Fragment into a flap. In most cases, the flap was removed by flap endonuclease 1 (FEN1), in a reaction required to remove initiator RNA in vivo. The nick left after flap removal could be sealed by DNA ligase I to complete Fragment joining. An alternative pathway involving FEN1 and the nuclease/helicase Dna2 has been proposed for flaps that become long enough to bind replication protein A (RPA). RPA binding can inhibit FEN1, but Dna2 can shorten RPA-bound flaps so that RPA dissociates. Recent reconstitution results indicated that Pif1 helicase, a known component of Fragment Processing, accelerated flap displacement, allowing the inhibitory action of RPA. In results presented here, Pif1 promoted DNA polymerase δ to displace strands that achieve a length to bind RPA, but also to be Dna2 substrates. Significantly, RPA binding to long flaps inhibited the formation of the final ligation products in the reconstituted system without Dna2. However, Dna2 reversed that inhibition to restore efficient ligation. These results suggest that the two-nuclease pathway is employed in cells to process long flap intermediates promoted by Pif1.
Yeon-soo Seo - One of the best experts on this subject based on the ideXlab platform.
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rad52 rad59 dependent recombination as a means to rectify faulty okazaki Fragment Processing
Journal of Biological Chemistry, 2014Co-Authors: Miju Lee, Chul-hwan Lee, Annie Albert Demin, Palinda Ruvan Munashingha, Tamir Amangyeld, Buki Kwon, Tim Formosa, Yeon-soo SeoAbstract:The correct removal of 5′-flap structures by Rad27 and Dna2 during Okazaki Fragment maturation is crucial for the stable maintenance of genetic materials and cell viability. In this study, we identified RAD52, a key recombination protein, as a multicopy suppressor of dna2-K1080E, a lethal helicase-negative mutant allele of DNA2 in yeasts. In contrast, the overexpression of Rad51, which works conjointly with Rad52 in canonical homologous recombination, failed to suppress the growth defect of the dna2-K1080E mutation, indicating that Rad52 plays a unique and distinct role in Okazaki Fragment metabolism. We found that the recombination-defective Rad52-QDDD/AAAA mutant did not rescue dna2-K1080E, suggesting that Rad52-mediated recombination is important for suppression. The Rad52-mediated enzymatic stimulation of Dna2 or Rad27 is not a direct cause of suppression observed in vivo, as both Rad52 and Rad52-QDDD/AAAA proteins stimulated the endonuclease activities of both Dna2 and Rad27 to a similar extent. The recombination mediator activity of Rad52 was dispensable for the suppression, whereas both the DNA annealing activity and its ability to interact with Rad59 were essential. In addition, we found that several cohesion establishment factors, including Rsc2 and Elg1, were required for the Rad52-dependent suppression of dna2-K1080E. Our findings suggest a novel Rad52/Rad59-dependent, but Rad51-independent recombination pathway that could ultimately lead to the removal of faulty flaps in conjunction with cohesion establishment factors.
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The Trans-autostimulatory Activity of Rad27 Suppresses dna2 Defects in Okazaki Fragment Processing
The Journal of biological chemistry, 2012Co-Authors: Palinda Ruvan Munashingha, Young-hoon Kang, Chul-hwan Lee, Yong-keol Shin, Tuan Anh Nguyen, Yeon-soo SeoAbstract:Dna2 and Rad27 (yeast Fen1) are the two endonucleases critical for Okazaki Fragment Processing during lagging strand DNA synthesis that have been shown to interact genetically and physically. In this study, we addressed the functional consequences of these interactions by examining whether purified Rad27 of Saccharomyces cerevisiae affects the enzymatic activity of Dna2 and vice versa. For this purpose, we constructed Rad27DA (catalytically defective enzyme with an Asp to Ala substitution at amino acid 179) and found that it significantly stimulated the endonuclease activity of wild type Dna2, but failed to do so with Dna2Δ405N that lacks the N-terminal 405 amino acids. This was an unexpected finding because dna2Δ405N cells were still partially suppressed by overexpression of rad27DA in vivo. Further analyses revealed that Rad27 is a trans-autostimulatory enzyme, providing an explanation why overexpression of Rad27, regardless of its catalytic activity, suppressed dna2 mutants as long as an endogenous wild type Rad27 is available. We found that the C-terminal 16-amino acid Fragment of Rad27, a highly polybasic region due to the presence of multiple positively charged lysine and arginine residues, was sufficient and necessary for the stimulation of both Rad27 and Dna2. Our findings provide further insight into how Dna2 and Rad27 jointly affect the Processing of Okazaki Fragments in eukaryotes.
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Dna2 on the road to Okazaki Fragment Processing and genome stability in eukaryotes.
Critical reviews in biochemistry and molecular biology, 2010Co-Authors: Young-hoon Kang, Chul-hwan Lee, Yeon-soo SeoAbstract:DNA replication is a primary mechanism for maintaining genome integrity, but it serves this purpose best by cooperating with other proteins involved in DNA repair and recombination. Unlike leading strand synthesis, lagging strand synthesis has a greater risk of faulty replication for several reasons: First, a significant part of DNA is synthesized by polymerase alpha, which lacks a proofreading function. Second, a great number of Okazaki Fragments are synthesized, processed and ligated per cell division. Third, the principal mechanism of Okazaki Fragment Processing is via generation of flaps, which have the potential to form a variety of structures in their sequence context. Finally, many proteins for the lagging strand interact with factors involved in repair and recombination. Thus, lagging strand DNA synthesis could be the best example of a converging place of both replication and repair proteins. To achieve the risky task with extraordinary fidelity, Okazaki Fragment Processing may depend on multiple layers of redundant, but connected pathways. An essential Dna2 endonuclease/helicase plays a pivotal role in Processing common structural intermediates that occur during diverse DNA metabolisms (e.g. lagging strand synthesis and telomere maintenance). Many roles of Dna2 suggest that the preemptive removal of long or structured flaps ultimately contributes to genome maintenance in eukaryotes. In this review, we describe the function of Dna2 in Okazaki Fragment Processing, and discuss its role in the maintenance of genome integrity with an emphasis on its functional interactions with other factors required for genome maintenance.
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The MPH1 gene of Saccharomyces cerevisiae functions in Okazaki Fragment Processing.
Journal of Biological Chemistry, 2009Co-Authors: Young-hoon Kang, Jerard Hurwitz, Min-jung Kang, Jeonghoon Kim, Chul-hwan Lee, Il-taeg Cho, Yeon-soo SeoAbstract:Saccharomyces cerevisiae MPH1 was first identified as a gene encoding a 3′ to 5′ DNA helicase, which when deleted leads to a mutator phenotype. In this study, we isolated MPH1 as a multicopy suppressor of the dna2K1080E helicase-negative lethal mutant. Purified Mph1 stimulated the endonuclease activities of both Fen1 and Dna2, which act faithfully in the Processing of Okazaki Fragments. This stimulation required neither ATP hydrolysis nor the helicase activity of Mph1. Multicopy expression of MPH1 also suppressed the temperature-sensitive growth defects in cells expressing dna2Δ405N, which lacks the N-terminal 405 amino acids of Dna2. However, Mph1 did not stimulate the endonuclease activity of the Dna2Δ405N mutant protein. The stimulation of Fen1 by Mph1 was limited to flap-structured substrates; Mph1 hardly stimulated the 5′ to 3′ exonuclease activity of Fen1. Mph1 binds to flap-structured substrate more efficiently than to nicked duplex structures, suggesting that the stimulatory effect of Mph1 is exerted through its binding to DNA substrates. In addition, we found that Mph1 reversed the inhibitory effects of replication protein A on Fen1 activity. Our biochemical and genetic data indicate that the in vivo suppression of Dna2 defects observed with both dna2K1080E and dna2Δ405N mutants occur via stimulation of Fen1 activity. These findings suggest that Mph1 plays an important, although not essential, role in Processing of Okazaki Fragments by facilitating the formation of ligatable nicks.
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In vivo and in vitro studies of Mgs1 suggest a link between genome instability and Okazaki Fragment Processing.
Nucleic Acids Research, 2005Co-Authors: Jeonghoon Kim, Young-hoon Kang, Min-jung Kang, Do-hyung Kim, Hyojin Kang, Gi-hyuck Ryu, Yeon-soo SeoAbstract:The non-essential MGS1 gene of Saccharomyces cerevisiae is highly conserved in eukaryotes and encodes an enzyme containing both DNAdependent ATPase and DNA annealing activities. MGS1 appears to function in post-replicational repair processes that contribute to genome stability. In this study, we identified MGS1 as a multicopy suppressor of the temperature-sensitive dna2D405N mutation, a DNA2 allele lacking the N-terminal 405 amino acid residues. Mgs1 stimulates the structure-specific nuclease activity of Rad27 (yeast Fen1 or yFen1) in an ATP-dependent manner. ATP binding but not hydrolysis was sufficient for the stimulatory effect of Mgs1, since non-hydrolyzable ATP analogs are as effective as ATP. Suppression of the temperaturesensitive growth defect of dna2D405N required the presence of a functional copy of RAD27, indicating that Mgs1 suppressed the dna2D405N mutation by increasing the activity of yFen1 (Rad27) in vivo. Our results provide in vivo and in vitro evidence that Mgs1 is involved in Okazaki Fragment Processing by modulating Fen1 activity. The data presented raise the possibility that the absence of MGS1 may impair the Processing of Okazaki Fragments, leading to genomic instability.
Michael R Lieber - One of the best experts on this subject based on the ideXlab platform.
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the fen 1 family of structure specific nucleases in eukaryotic dna replication recombination and repair
BioEssays, 1997Co-Authors: Michael R LieberAbstract:Unlike the most well-characterized prokaryotic polymerase, E. Coli DNA pol I, none of the eukaryotic polymerases have their own 5′ to 3′ exonuclease domain for nick translation and Okazaki Fragment Processing. In eukaryotes, FEN-1 is an endo-and exonuclease that carries out this function independently of the polymerase molecules. Only seven nucleases have been cloned from multicellular eukaryotic cells. Among these, FEN-1 is intriguing because it has complex structural preferences; specifically, it cleaves at branched DNA structures. The cloning of FEN-1 permitted establishment of the first eukaryotic nuclease family, predicting that S. cerevisiae RAD2 (S. pombe Rad13) and its mammalian homolog, XPG, would have similar structural specficity. The FEN-1 nuclease family includes several similar enzymes encoded by bacteriophages. The crystal structures of two enzymes in the FEN-1 nuclease family have been solved and they provide a structural basis for the interesting steric requirements of FEN-1 substrates. Because of their unique structural specificities, FEN-1 and its family members have important roles in DNA replication, repair and, potentially, recombination. Recently, FEN-1 was found to specifically associate with PCNA, explaining some aspects of FEN-1 function during DNA replication and potentially in DNA repair.
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the fen 1 family of structure specific nucleases in eukaryotic dna replication recombination and repair
BioEssays, 1997Co-Authors: Michael R LieberAbstract:Unlike the most well-characterized prokaryotic polymerase, E. Coli DNA pol I, none of the eukaryotic polymerases have their own 5′ to 3′ exonuclease domain for nick translation and Okazaki Fragment Processing. In eukaryotes, FEN-1 is an endo-and exonuclease that carries out this function independently of the polymerase molecules. Only seven nucleases have been cloned from multicellular eukaryotic cells. Among these, FEN-1 is intriguing because it has complex structural preferences; specifically, it cleaves at branched DNA structures. The cloning of FEN-1 permitted establishment of the first eukaryotic nuclease family, predicting that S. cerevisiae RAD2 (S. pombe Rad13) and its mammalian homolog, XPG, would have similar structural specficity. The FEN-1 nuclease family includes several similar enzymes encoded by bacteriophages. The crystal structures of two enzymes in the FEN-1 nuclease family have been solved and they provide a structural basis for the interesting steric requirements of FEN-1 substrates. Because of their unique structural specificities, FEN-1 and its family members have important roles in DNA replication, repair and, potentially, recombination. Recently, FEN-1 was found to specifically associate with PCNA, explaining some aspects of FEN-1 function during DNA replication and potentially in DNA repair.
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lagging strand dna synthesis at the eukaryotic replication fork involves binding and stimulation of fen 1 by proliferating cell nuclear antigen
Journal of Biological Chemistry, 1995Co-Authors: Xiangyang Li, Michael R Lieber, Jun Li, John J Harrington, Peter M J BurgersAbstract:Abstract The 5′ 3′-exonuclease domain of Escherichia coli DNA polymerase I is required for the completion of lagging strand DNA synthesis, and yet this domain is not present in any of the eukaryotic DNA polymerases. Recently, the gene encoding the functional and evolutionary equivalent of this 5′ 3′-exonuclease domain has been identified. It is called FEN-1 in mouse and human cells and RTH1 in Saccharomyces cerevisiae. This 42-kDa enzyme is required for Okazaki Fragment Processing. Here we report that FEN-1 physically interacts with proliferating cell nuclear antigen (PCNA), the processivity factor for DNA polymerases and . Through protein-protein interactions, PCNA focuses FEN-1 on branched DNA substrates (flap structures) and on nicked DNA substrates, thereby stimulating its activity 10-50-fold but only if PCNA can functionally assemble as a toroidal trimer around the DNA. This interaction is important in the physical orchestration of lagging strand synthesis and may have implications for how PCNA stimulates other members of the FEN-1 nuclease family in a broad range of DNA metabolic transactions.
Peter M J Burgers - One of the best experts on this subject based on the ideXlab platform.
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An Alternative Pathway for Okazaki Fragment Processing RESOLUTION OF FOLD-BACK FLAPS BY Pif1 HELICASE
Journal of Biological Chemistry, 2010Co-Authors: Jason E. Pike, Peter M J Burgers, Judith L. Campbell, Ryan A. Henry, Robert A BambaraAbstract:Two pathways have been proposed for eukaryotic Okazaki Fragment RNA primer removal. Results presented here provide evidence for an alternative pathway. Primer extension by DNA polymerase δ (pol δ) displaces the downstream Fragment into an RNA-initiated flap. Most flaps are cleaved by flap endonuclease 1 (FEN1) while short, and the remaining nicks joined in the first pathway. A small fraction escapes immediate FEN1 cleavage and is further lengthened by Pif1 helicase. Long flaps are bound by replication protein A (RPA), which inhibits FEN1. In the second pathway, Dna2 nuclease cleaves an RPA-bound flap and displaces RPA, leaving a short flap for FEN1. Pif1 flap lengthening creates a requirement for Dna2. This relationship should not have evolved unless Pif1 had an important role in Fragment Processing. In this study, biochemical reconstitution experiments were used to gain insight into this role. Pif1 did not promote synthesis through GC-rich sequences, which impede strand displacement. Pif1 was also unable to open fold-back flaps that are immune to cleavage by either FEN1 or Dna2 and cannot be bound by RPA. However, Pif1 working with pol δ readily unwound a full-length Okazaki Fragment initiated by a fold-back flap. Additionally, a fold-back in the template slowed pol δ synthesis, so that the Fragment could be removed before ligation to the lagging strand. These results suggest an alternative pathway in which Pif1 removes Okazaki Fragments initiated by fold-back flaps in vivo.
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pif1 helicase lengthens some okazaki Fragment flaps necessitating dna2 nuclease helicase action in the two nuclease Processing pathway
Journal of Biological Chemistry, 2009Co-Authors: Jason E. Pike, Peter M J Burgers, Judith L. Campbell, Robert A BambaraAbstract:We have developed a system to reconstitute all of the proposed steps of Okazaki Fragment Processing using purified yeast proteins and model substrates. DNA polymerase δ was shown to extend an upstream Fragment to displace a downstream Fragment into a flap. In most cases, the flap was removed by flap endonuclease 1 (FEN1), in a reaction required to remove initiator RNA in vivo. The nick left after flap removal could be sealed by DNA ligase I to complete Fragment joining. An alternative pathway involving FEN1 and the nuclease/helicase Dna2 has been proposed for flaps that become long enough to bind replication protein A (RPA). RPA binding can inhibit FEN1, but Dna2 can shorten RPA-bound flaps so that RPA dissociates. Recent reconstitution results indicated that Pif1 helicase, a known component of Fragment Processing, accelerated flap displacement, allowing the inhibitory action of RPA. In results presented here, Pif1 promoted DNA polymerase δ to displace strands that achieve a length to bind RPA, but also to be Dna2 substrates. Significantly, RPA binding to long flaps inhibited the formation of the final ligation products in the reconstituted system without Dna2. However, Dna2 reversed that inhibition to restore efficient ligation. These results suggest that the two-nuclease pathway is employed in cells to process long flap intermediates promoted by Pif1.
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lagging strand dna synthesis at the eukaryotic replication fork involves binding and stimulation of fen 1 by proliferating cell nuclear antigen
Journal of Biological Chemistry, 1995Co-Authors: Xiangyang Li, Michael R Lieber, Jun Li, John J Harrington, Peter M J BurgersAbstract:Abstract The 5′ 3′-exonuclease domain of Escherichia coli DNA polymerase I is required for the completion of lagging strand DNA synthesis, and yet this domain is not present in any of the eukaryotic DNA polymerases. Recently, the gene encoding the functional and evolutionary equivalent of this 5′ 3′-exonuclease domain has been identified. It is called FEN-1 in mouse and human cells and RTH1 in Saccharomyces cerevisiae. This 42-kDa enzyme is required for Okazaki Fragment Processing. Here we report that FEN-1 physically interacts with proliferating cell nuclear antigen (PCNA), the processivity factor for DNA polymerases and . Through protein-protein interactions, PCNA focuses FEN-1 on branched DNA substrates (flap structures) and on nicked DNA substrates, thereby stimulating its activity 10-50-fold but only if PCNA can functionally assemble as a toroidal trimer around the DNA. This interaction is important in the physical orchestration of lagging strand synthesis and may have implications for how PCNA stimulates other members of the FEN-1 nuclease family in a broad range of DNA metabolic transactions.
