The Experts below are selected from a list of 324 Experts worldwide ranked by ideXlab platform
Anindya Dutta - One of the best experts on this subject based on the ideXlab platform.
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The Evolutionarily Conserved Zinc Finger Motif in the Largest Subunit of Human Replication Protein A Is Required for DNA
2013Co-Authors: Anindya DuttaAbstract:The largest subunit of the replication protein A (RPA) contains an evolutionarily conserved zinc finger motif that lies outside of the domains required for binding to single-stranded DNA or forming the RPA holocomplex. In previous studies, we showed that a point mutation in this motif (RPA m) cannot support SV40 DNA replication. We have now investigated the role of this motif in several steps of DNA replication and in two DNA repair pathways. RPA m associates with T antigen, assists the unwinding of double-stranded DNA at an origin of replication, stimulates DNA polymerases � and �, and supports the formation of the initial short Okazaki Fragments. However, the synthesis of a leading strand and later Okazaki Fragments is impaired. In contrast, RPA m can function well during the incision step of nucleotid
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the evolutionarily conserved zinc finger motif in the largest subunit of human replication protein a is required for dna replication and mismatch repair but not for nucleotide excision repair
Journal of Biological Chemistry, 1998Co-Authors: Mahmud K K Shivji, Clark C Chen, Richard D Kolodner, Richard D Wood, Anindya DuttaAbstract:Abstract The largest subunit of the replication protein A (RPA) contains an evolutionarily conserved zinc finger motif that lies outside of the domains required for binding to single-stranded DNA or forming the RPA holocomplex. In previous studies, we showed that a point mutation in this motif (RPAm) cannot support SV40 DNA replication. We have now investigated the role of this motif in several steps of DNA replication and in two DNA repair pathways. RPAm associates with T antigen, assists the unwinding of double-stranded DNA at an origin of replication, stimulates DNA polymerases α and δ, and supports the formation of the initial short Okazaki Fragments. However, the synthesis of a leading strand and later Okazaki Fragments is impaired. In contrast, RPAm can function well during the incision step of nucleotide excision repair and in a full repair synthesis reaction, with either UV-damaged or cisplatin-adducted DNA. Two deletion mutants of the Rpa1 subunit (eliminating amino acids 1–278 or 222–411) were not functional in nucleotide excision repair. We report for the first time that wild type RPA is required for a mismatch repair reaction in vitro. Neither the deletion mutants nor RPAm can support this reaction. Therefore, the zinc finger of the largest subunit of RPA is required for a function that is essential for DNA replication and mismatch repair but not for nucleotide excision repair.
Chengyao Chen - One of the best experts on this subject based on the ideXlab platform.
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a multifunctional dna polymerase i involves in the maturation of Okazaki Fragments during the lagging strand dna synthesis in helicobacter pylori
FEBS Journal, 2020Co-Authors: Yiwen Cheng, Chengyao ChenAbstract:Helicobacter pylori is the most infectious human pathogen that causes gastritis, peptic ulcers and stomach cancer. H. pylori DNA polymerase I (HpPol I) is found to be essential for the viability of H. pylori, but its intrinsic property and attribution to the H. pylori DNA replication remain unclear. HpPol I contains a 5'→3' exonuclease (5'-Exo) and DNA polymerase (Pol) domain, respectively, but lacks a 3'→5' exonuclease, or error proofreading activity. In this study, we characterized the 5'-Exo and Pol functions of HpPol I and found that HpPol I is a multifunctional protein displaying DNA nick translation, strand-displacement synthesis, RNase H-like, structure-specific endonuclease and exonuclease activities. In the in vitro DNA replication assay, we further demonstrated that the 5'-Exo and Pol domains of HpPol I can cooperate to fill in the DNA gap, remove the unwanted RNA primer from a RNA/DNA hybrid and create a ligatable nick for the DNA ligase A of H. pylori to restore the normal duplex DNA. Altogether, our study suggests that the two catalytic domains of HpPol I may synergistically play an important role in the maturation of Okazaki Fragments during the lagging-strand DNA synthesis in H. pylori. Like the functions of DNA polymerase I in Escherichia coli, HpPol I may involve in both DNA replication and repair in H. pylori.
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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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.
Michelle M. Spiering - 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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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.
Duncan J. Smith - One of the best experts on this subject based on the ideXlab platform.
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hold on tight lagging strand dna polymerases synthesize multiple Okazaki Fragments without letting go
Molecular Cell, 2020Co-Authors: Vanessa Kellner, Duncan J. SmithAbstract:Kapadia et al. (2020) use an innovative single-molecule imaging approach in yeast cells to measure chromatin association of individual replisome subunits, thereby challenging the notion that lagging-strand DNA polymerases frequently dissociate from replisomes during DNA replication in vivo.
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Processing of eukaryotic Okazaki Fragments by redundant nucleases can be uncoupled from ongoing DNA replication in vivo.
Nucleic Acids Research, 2018Co-Authors: Malik Kahli, Joseph Osmundson, Rani Yeung, Duncan J. SmithAbstract:Prior to ligation, each Okazaki fragment synthesized on the lagging strand in eukaryotes must be nucleolytically processed. Nuclease cleavage takes place in the context of 5' flap structures generated via strand-displacement synthesis by DNA polymerase delta. At least three DNA nucleases: Rad27 (Fen1), Dna2 and Exo1, have been implicated in processing Okazaki fragment flaps. However, neither the contributions of individual nucleases to lagging-strand synthesis nor the structure of the DNA intermediates formed in their absence have been fully defined in vivo. By conditionally depleting lagging-strand nucleases and directly analyzing Okazaki Fragments synthesized in vivo in Saccharomyces cerevisiae, we conduct a systematic evaluation of the impact of Rad27, Dna2 and Exo1 on lagging-strand synthesis. We find that Rad27 processes the majority of lagging-strand flaps, with a significant additional contribution from Exo1 but not from Dna2. When nuclease cleavage is impaired, we observe a reduction in strand-displacement synthesis as opposed to the widespread generation of long Okazaki fragment 5' flaps, as predicted by some models. Further, using cell cycle-restricted constructs, we demonstrate that both the nucleolytic processing and the ligation of Okazaki Fragments can be uncoupled from DNA replication and delayed until after synthesis of the majority of the genome is complete.
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Detection and Sequencing of Okazaki Fragments in S. cerevisiae
Methods of Molecular Biology, 2015Co-Authors: Duncan J. Smith, Tejas Yadav, Iestyn WhitehouseAbstract:We have previously demonstrated that lagging-strand synthesis in budding yeast is coupled with chromatin assembly on newly synthesized DNA. Using a strain of S. cerevisiae in which DNA ligase I can be conditionally depleted, we can enrich and purify Okazaki Fragments. We delineate a method to extract, end label, and visualize Okazaki Fragments using denaturing agarose gel electrophoresis. Furthermore, we describe an ion-exchange chromatographic method for purification of Fragments and preparation of strand-specific sequencing libraries. Deep sequencing of Okazaki Fragments generates a comprehensive, genomic map of DNA synthesis, starting from a single asynchronous culture. Altogether this approach represents a tractable system to investigate key aspects of DNA replication and chromatin assembly.
