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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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Rad52/Rad59-dependent recombination as a means to rectify faulty Okazaki Fragment processing.
The 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, Chul-hwan Lee, Young-hoon Kang, 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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The Trans-autostimulatory Activity of Rad27 Suppresses dna2 Defects in Okazaki Fragment Processing
Journal of Biological Chemistry, 2012Co-Authors: Palinda Ruvan Munashingha, Chul-hwan Lee, Young-hoon Kang, Yong-keol Shin, Tuan Anh Nguyen, Yeon Soo SeoAbstract:Abstract 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 since dna2Δ405N cells were still partially suppressed by overexpression of rad27DA in vivo. Further analyses revealed that Rad27 is a trans-auto stimulatory 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.
Robert A Bambara - One of the best experts on this subject based on the ideXlab platform.
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Okazaki Fragment Metabolism
Cold Spring Harbor Perspectives in Biology, 2013Co-Authors: Lata Balakrishnan, Robert A BambaraAbstract:Replication of cellular chromosomal DNA is initiated by the multienzyme replisome machinery, which unwinds the DNA helix to create a replication fork. The antiparallel structure of double-helical DNA and the 3′ end extension specificity of all DNA polymerases confine the mechanisms that can be used by the cell for DNA duplication. One copied strand, called leading, can conveniently be extended in a continuous manner in the same direction that the helix must open to allow exposure of templates for polymerization. The other, or lagging strand, must be periodically extended away from the opening helix. This can only be accomplished if the strand is made discontinuously (Kornberg and Baker 1992). The strand is synthesized in short segments, named Okazaki Fragments, after their discoverer (Sakabe and Okazaki 1966; Okazaki et al. 1968) and the segments are then joined. This requirement has two fundamental consequences: (1) The lagging strand must have evolved priming and Fragment joining mechanisms involving many additional steps and reactions than needed for leading-strand extension. (2) Mechanisms of lagging-strand replication must have developed means of avoiding mutagenesis while handling the necessary strand manipulations.
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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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An Alternative Pathway for Okazaki Fragment Processing
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 (pol ) 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 RPAbound 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 readily unwound a full-length Okazaki Fragment initiated by a fold-back flap. Additionally, a foldback in the template slowed pol 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.
Peter M J Burgers - One of the best experts on this subject based on the ideXlab platform.
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Solution to the 50-year-old Okazaki-Fragment problem
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Peter M J BurgersAbstract:The antiparallel structure of double-stranded DNA, together with the known 5′→3′ directionality of DNA polymerases, necessitates that the two DNA strands are replicated in opposite directions. The leading strand is synthesized in the same direction as the replication fork, whereas the lagging strand is replicated in the opposite direction. In a 1968 paper in PNAS, Reiji and Tsuneko Okazaki and colleagues (1) proposed that the lagging strand is replicated discontinuously in the form of small Fragments that subsequently are matured into one continuous strand. A review of the current state of molecular biology published that year (2) named these small Fragments “Okazaki Fragments,” as they have been called since. The seminal studies of Okazaki et al. (1) generated the textbook model of the semidiscontinuous replication fork, with a continuous leading strand and a discontinuous lagging strand. However, their original experimental results were not in accord with this model, and their studies suggested that all nascent DNA Fragments are small. Was the leading strand also synthesized discontinuously, as was depicted in figure 1 of ref. 1 (Fig. 1 C )? Fifty years after the landmark paper, this question has been answered by Cronan et al. (3), who show that the leading strand is indeed replicated continuously. However, this strand is Fragmented due to ribonucleotide excision repair (RER) (4, 5). RER removes genomic ribonucleotides that are erroneously inserted by replicative DNA polymerases (Fig. 2 A ). Fig. 1. ( A – D ) Models for the possible structure and reaction in the replicating region of DNA. Reprinted with permission from ref. 1. Fig. 2. ( A ) DNA incision intermediates in the BER of a damaged or unnatural base (X), or the RER of a genomic ribonucleotide. Only repair on the leading strand, which leads to its Fragmentation, is shown. ( B ) Size distribution, in a formamide-urea-sucrose gradient, of single-stranded replication intermediates. … [↵][1]1Email: burgers{at}wustl.edu. [1]: #xref-corresp-1-1
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Sequential switching of binding partners on PCNA during in vitro Okazaki Fragment maturation
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Daniel Dovrat, Peter M J Burgers, Joseph L. Stodola, Amir AharoniAbstract:The homotrimeric sliding clamp proliferating cell nuclear antigen (PCNA) mediates Okazaki Fragment maturation through tight coordination of the activities of DNA polymerase δ (Pol δ), flap endonuclease 1 (FEN1) and DNA ligase I (Lig1). Little is known regarding the mechanism of partner switching on PCNA and the involvement of PCNA9s three binding sites in coordinating such processes. To shed new light on PCNA-mediated Okazaki Fragment maturation, we developed a novel approach for the generation of PCNA heterotrimers containing one or two mutant monomers that are unable to bind and stimulate partners. These heterotrimers maintain the native oligomeric structure of PCNA and exhibit high stability under various conditions. Unexpectedly, we found that PCNA heterotrimers containing only one functional binding site enable Okazaki Fragment maturation by efficiently coordinating the activities of Pol δ, FEN1, and Lig1. The efficiency of switching between partners on PCNA was not significantly impaired by limiting the number of available binding sites on the PCNA ring. Our results provide the first direct evidence, to our knowledge, that simultaneous binding of multiple partners to PCNA is unnecessary, and if it occurs, does not provide significant functional advantages for PCNA-mediated Okazaki Fragment maturation in vitro. In contrast to the “toolbelt” model, which was demonstrated for bacterial and archaeal sliding clamps, our results suggest a mechanism of sequential switching of partners on the eukaryotic PCNA trimer during DNA replication and repair.
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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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An Alternative Pathway for Okazaki Fragment Processing
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 (pol ) 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 RPAbound 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 readily unwound a full-length Okazaki Fragment initiated by a fold-back flap. Additionally, a foldback in the template slowed pol 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.
Sung-ho Bae - One of the best experts on this subject based on the ideXlab platform.
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Involvement of Vts1, a structure-specific RNA-binding protein, in Okazaki Fragment processing in yeast
Nucleic Acids Research, 2009Co-Authors: Chul-hwan Lee, Young-hoon Kang, Jeong-hoon Kim, Do-hyung Kim, Yong-keol Shin, Tuan Anh Nguyen, Min-jung Kang, Thi Thu Huong Phung, Jae Seok Bae, Sung-ho BaeAbstract:The non-essential VTS1 gene of Saccharomyces cerevisiae is highly conserved in eukaryotes and encodes a sequence- and structure-specific RNA-binding protein. The Vts1 protein has been implicated in post-transcriptional regulation of a specific set of mRNAs that contains its-binding site at their 3′-untranslated region. In this study, we identified VTS1 as a multi-copy suppressor of dna2-K1080E, a lethal mutant allele of DNA2 that lacks DNA helicase activity. The suppression was allele-specific, since overexpression of Vts1 did not suppress the temperature-dependent growth defects of dna2Δ405N devoid of the N-terminal 405-amino-acid residues. Purified recombinant Vts1 stimulated the endonuclease activity of wild-type Dna2, but not the endonuclease activity of Dna2Δ405N, indicating that the activation requires the N-terminal domain of Dna2. Stimulation of Dna2 endonuclease activity by Vts1 appeared to be the direct cause of suppression, since the multi-copy expression of Dna2-K1080E suppressed the lethality observed with its single-copy expression. We found that vts1Δ dna2Δ405N and vts1Δdna2-7 double mutant cells displayed synergistic growth defects, in support of a functional interaction between two genes. Our results provide both in vivo and in vitro evidence that Vts1 is involved in lagging strand synthesis by modulating the Dna2 endonuclease activity that plays an essential role in Okazaki Fragment processing.
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Coupling of DNA helicase and endonuclease activities of yeast Dna2 facilitates Okazaki Fragment processing.
Journal of Biological Chemistry, 2002Co-Authors: Sung-ho Bae, Hoyoung Kang, Hee-dai Kim, Dong-wook Kim, Ji-young Kim, Jeong-hoon Kim, Do-hyung Kim, Yeon Soo SeoAbstract:Saccharomyces cerevisiae Dna2 possesses both helicase and endonuclease activities. Its endonuclease activity is essential and well suited to remove RNA-DNA primers of Okazaki Fragments. In contrast, its helicase activity, although required for optimal growth, is not essential when the rate of cell growth is reduced. These findings suggest that DNA unwinding activity of Dna2 plays an auxiliary role in Okazaki Fragment processing. To address this issue, we examined whether the Dna2 helicase activity influenced its intrinsic endonuclease activity using two mutant proteins, Dna2D657A and Dna2K1080E, which contain only helicase or endonuclease activity, respectively. Experiments performed with a mixture of Dna2D657A and Dna2K1080E enzymes revealed that cleavage of a single-stranded DNA by endonuclease activity of Dna2 occurs while the enzyme translocates along the substrate. In addition, DNA unwinding activity efficiently removed the secondary structure formed in the flap structure, which was further aided by replication protein A. Our results suggest that the Dna2 unwinding activity plays a role in facilitating the removal of the flap DNA by its intrinsic endonuclease activity.
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characterization of the enzymatic properties of the yeast dna2 helicase endonuclease suggests a new model for Okazaki Fragment processing
Journal of Biological Chemistry, 2000Co-Authors: Sung-ho Bae, Yeon Soo SeoAbstract:Abstract The Saccharomyces cerevisiae Dna2, which contains single-stranded DNA-specific endonuclease activity, interacts genetically and physically with Fen-1, a structure-specific endonuclease implicated in Okazaki Fragment maturation during lagging strand synthesis. In this report, we investigated the properties of the Dna2 helicase/endonuclease activities in search of their in vivo physiological functions in eukaryotes. We found that the Dna2 helicase activity translocates in the 5′ to 3′ direction and uses DNA with free ends as the preferred substrate. Furthermore, the endonucleolytic cleavage activity of Dna2 was markedly stimulated by the presence of an RNA segment at the 5′-end of single-stranded DNA and occurred within the DNA, ensuring the complete removal of the initiator RNA segment on the Okazaki Fragment. In addition, we demonstrated that the removal of pre-existing initiator 5′-terminal RNA segments depended on a displacement reaction carried out during the DNA polymerase δ-catalyzed elongation of the upstream Okazaki Fragments. These properties indicate that Dna2 is well suited to remove the primer RNA on the Okazaki Fragment. Based op this information, we propose a new model in which Dna2 plays a direct role in Okazaki Fragment maturation in conjunction with Fen-1.
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Characterization of the enzymatic properties of the yeast dna2 Helicase/endonuclease suggests a new model for Okazaki Fragment processing.
Journal of Biological Chemistry, 2000Co-Authors: Sung-ho Bae, Yeon Soo SeoAbstract:Abstract The Saccharomyces cerevisiae Dna2, which contains single-stranded DNA-specific endonuclease activity, interacts genetically and physically with Fen-1, a structure-specific endonuclease implicated in Okazaki Fragment maturation during lagging strand synthesis. In this report, we investigated the properties of the Dna2 helicase/endonuclease activities in search of their in vivo physiological functions in eukaryotes. We found that the Dna2 helicase activity translocates in the 5′ to 3′ direction and uses DNA with free ends as the preferred substrate. Furthermore, the endonucleolytic cleavage activity of Dna2 was markedly stimulated by the presence of an RNA segment at the 5′-end of single-stranded DNA and occurred within the DNA, ensuring the complete removal of the initiator RNA segment on the Okazaki Fragment. In addition, we demonstrated that the removal of pre-existing initiator 5′-terminal RNA segments depended on a displacement reaction carried out during the DNA polymerase δ-catalyzed elongation of the upstream Okazaki Fragments. These properties indicate that Dna2 is well suited to remove the primer RNA on the Okazaki Fragment. Based op this information, we propose a new model in which Dna2 plays a direct role in Okazaki Fragment maturation in conjunction with Fen-1.
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Genetic analyses of Schizosaccharomyces pombe dna2(+) reveal that dna2 plays an essential role in Okazaki Fragment metabolism.
Genetics, 2000Co-Authors: Hoyoung Kang, Sung-ho Bae, Eunjoo Choi, Kyoung-hwa Lee, Byung-soo Gim, Hee-dai Kim, Chankyu Park, Stuart A. Macneill, Yeon Soo SeoAbstract:In this report, we investigated the phenotypes caused by temperature-sensitive (ts) mutant alleles of dna2(+) of Schizosaccharomyces pombe, a homologue of DNA2 of budding yeast, in an attempt to further define its function in vivo with respect to lagging-strand synthesis during the S-phase of the cell cycle. At the restrictive temperature, dna2 (ts) cells arrested at late S-phase but were unaffected in bulk DNA synthesis. Moreover, they exhibited aberrant mitosis when combined with checkpoint mutations, in keeping with a role for Dna2 in Okazaki Fragment maturation. Similarly, spores in which dna2(+) was disrupted duplicated their DNA content during germination and also arrested at late S-phase. Inactivation of dna2(+) led to chromosome Fragmentation strikingly similar to that seen when cdc17(+), the DNA ligase I gene, is inactivated. The temperature-dependent lethality of dna2 (ts) mutants was suppressed by overexpression of genes encoding subunits of polymerase delta (cdc1(+) and cdc27(+)), DNA ligase I (cdc17(+)), and Fen-1 (rad2(+)). Each of these gene products plays a role in the elongation or maturation of Okazaki Fragments. Moreover, they all interacted with S. pombe Dna2 in a yeast two-hybrid assay, albeit to different extents. On the basis of these results, we conclude that dna2(+) plays a direct role in the Okazaki Fragment elongation and maturation. We propose that dna2(+) acts as a central protein to form a complex with other proteins required to coordinate the multienzyme process for Okazaki Fragment elongation and maturation.
Stephen J. Benkovic - One of the best experts on this subject based on the ideXlab platform.
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rna primer primase complexes serve as the signal for polymerase recycling and Okazaki Fragment initiation in t4 phage dna replication
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Michelle M. Spiering, Philip Hanoian, Swathi Gannavaram, Stephen J. BenkovicAbstract:The opposite strand polarity of duplex DNA necessitates that the leading strand is replicated continuously whereas the lagging strand is replicated in discrete segments known as Okazaki Fragments. The lagging-strand polymerase sometimes recycles to begin the synthesis of a new Okazaki Fragment before finishing the previous Fragment, creating a gap between the Okazaki Fragments. The mechanism and signal that initiate this behavior—that is, the signaling mechanism—have not been definitively identified. We examined the role of RNA primer–primase complexes left on the lagging ssDNA from primer synthesis in initiating early lagging-strand polymerase recycling. We show for the T4 bacteriophage DNA replication system that primer–primase complexes have a residence time similar to the timescale of Okazaki Fragment synthesis and the ability to block a holoenzyme synthesizing DNA and stimulate the dissociation of the holoenzyme to trigger polymerase recycling. The collision with primer–primase complexes triggering the early termination of Okazaki Fragment synthesis has distinct advantages over those previously proposed because this signal requires no transmission to the lagging-strand polymerase through protein or DNA interactions, the mechanism for rapid dissociation of the holoenzyme is always collision, and no unique characteristics need to be assigned to either identical polymerase in the replisome. We have modeled repeated cycles of Okazaki Fragment initiation using a collision with a completed Okazaki Fragment or primer–primase complexes as the recycling mechanism. The results reproduce experimental data, providing insights into events related to Okazaki Fragment initiation and the overall functioning of DNA replisomes.
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RNA primer–primase complexes serve as the signal for polymerase recycling and Okazaki Fragment initiation in T4 phage DNA replication
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Michelle M. Spiering, Philip Hanoian, Swathi Gannavaram, Stephen J. BenkovicAbstract:The opposite strand polarity of duplex DNA necessitates that the leading strand is replicated continuously whereas the lagging strand is replicated in discrete segments known as Okazaki Fragments. The lagging-strand polymerase sometimes recycles to begin the synthesis of a new Okazaki Fragment before finishing the previous Fragment, creating a gap between the Okazaki Fragments. The mechanism and signal that initiate this behavior—that is, the signaling mechanism—have not been definitively identified. We examined the role of RNA primer–primase complexes left on the lagging ssDNA from primer synthesis in initiating early lagging-strand polymerase recycling. We show for the T4 bacteriophage DNA replication system that primer–primase complexes have a residence time similar to the timescale of Okazaki Fragment synthesis and the ability to block a holoenzyme synthesizing DNA and stimulate the dissociation of the holoenzyme to trigger polymerase recycling. The collision with primer–primase complexes triggering the early termination of Okazaki Fragment synthesis has distinct advantages over those previously proposed because this signal requires no transmission to the lagging-strand polymerase through protein or DNA interactions, the mechanism for rapid dissociation of the holoenzyme is always collision, and no unique characteristics need to be assigned to either identical polymerase in the replisome. We have modeled repeated cycles of Okazaki Fragment initiation using a collision with a completed Okazaki Fragment or primer–primase complexes as the recycling mechanism. The results reproduce experimental data, providing insights into events related to Okazaki Fragment initiation and the overall functioning of DNA replisomes.
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stability of the human polymerase δ holoenzyme and its implications in lagging strand dna synthesis
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Mark Hedglin, Binod Pandey, Stephen J. BenkovicAbstract:In eukaryotes, DNA polymerase δ (pol δ) is responsible for replicating the lagging strand template and anchors to the proliferating cell nuclear antigen (PCNA) sliding clamp to form a holoenzyme. The stability of this complex is integral to every aspect of lagging strand replication. Most of our understanding comes from Saccharomyces cerevisae where the extreme stability of the pol δ holoenzyme ensures that every nucleobase within an Okazaki Fragment is faithfully duplicated before dissociation but also necessitates an active displacement mechanism for polymerase recycling and exchange. However, the stability of the human pol δ holoenzyme is unknown. We designed unique kinetic assays to analyze the processivity and stability of the pol δ holoenzyme. Surprisingly, the results indicate that human pol δ maintains a loose association with PCNA while replicating DNA. Such behavior has profound implications on Okazaki Fragment synthesis in humans as it limits the processivity of pol δ on undamaged DNA and promotes the rapid dissociation of pol δ from PCNA on stalling at a DNA lesion.
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Insights into Okazaki Fragment Synthesis by the T4 Replisome THE FATE OF LAGGING-STRAND HOLOENZYME COMPONENTS AND THEIR INFLUENCE ON Okazaki Fragment SIZE
Journal of Biological Chemistry, 2013Co-Authors: Danqi Chen, Hongjun Yue, Michelle M. Spiering, Stephen J. BenkovicAbstract:Abstract In this study, we employed a circular replication substrate with a low priming site frequency (1 site/1.1 kb) to quantitatively examine the size distribution and formation pattern of Okazaki Fragments. Replication reactions by the T4 replisome on this substrate yielded a patterned series of Okazaki Fragments whose size distribution shifted through collision and signaling mechanisms as the gp44/62 clamp loader levels changed but was insensitive to changes in the gp43 polymerase concentration, as expected for a processive, recycled lagging-strand polymerase. In addition, we showed that only one gp45 clamp is continuously associated with the replisome and that no additional clamps accumulate on the DNA, providing further evidence that the clamp departs, whereas the polymerase is recycled upon completion of an Okazaki Fragment synthesis cycle. We found no support for the participation of a third polymerase in Okazaki Fragment synthesis.
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Dissociation of bacteriophage T4 DNa polymerase and its processivity clamp after completion of Okazaki Fragment synthesis
Biochemistry, 1997Co-Authors: Theodore E. Carver, Daniel J. Sexton, Stephen J. BenkovicAbstract:The mechanism of bacteriophage T4 DNA polymerase (gp43) and clamp (gp45) protein dissociation from the holoenzyme·DNA complex was investigated under conditions simulating the environment encountered upon completion of an Okazaki Fragment. Lagging strand DNA synthesis was approximated using a synthetic construct comprised of a doubly biotinylated, streptavidin-bound 62-mer DNA template, paired with complementary primers to generate an internal 12-base gap where the 5‘-end primer contained either a 5‘-OH (DNA primer) or a 5‘-triphosphate (RNA primer) group. Rapid kinetic measurements revealed that upon encountering the blocking primer, the holoenzyme either dissociates from DNA (∼40%) or strand-displaces the blocking strand (∼60%). The two blocking oligonucleotides (DNA or RNA) induce a 30−50-fold increase in the rate of holoenzyme dissociation, with both polymerase and clamp proteins dissociating simultaneously. Inhibition of ATP hydrolysis by ATP-γ-S did not have a measurable effect upon holoenzyme dissoc...
