The Wonders of Flap Endonucleases: Structure, Function, Mechanism and Regulation

Finger, L. David; Atack, John M.; Tsutakawa, Susan E.; Classen, Scott; Tainer, John A.; Grasby, Jane A.; Shen, Binghui

doi:10.1007/978-94-007-4572-8_16

Cited by 49 publications

(66 citation statements)

References 102 publications

(182 reference statements)

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“…If the exonuclease pathway plays a role in removing the RNA-DNA primers, then the DNA exonucleases responsible for hydrolyzing the DNA portion of the RNA-DNA primers have not been definitively identified. In yeast, Dna2 and Fen1 do not appear to participate in the exonuclease pathway because yeast Dna2 and Fen1 lack or have very weak double-stranded DNA exonuclease activity (29,30). Regarding the flap pathway, direct in vivo evidence demonstrating that the RNA-DNA primers are displaced to form flap structures and that the flap structures are subsequently cleaved by Dna2 and Fen1 is lacking.…”

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confidence: 99%

Direct Visualization of RNA-DNA Primer Removal from Okazaki Fragments Provides Support for Flap Cleavage and Exonucleolytic Pathways in Eukaryotic Cells

Liu

Wang

et al. 2017

Journal of Biological Chemistry

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Edited by Patrick SungDuring DNA replication in eukaryotic cells, short singlestranded DNA segments known as Okazaki fragments are first synthesized on the lagging strand. The Okazaki fragments originate from ϳ35-nucleotide-long RNA-DNA primers. After Okazaki fragment synthesis, these primers must be removed to allow fragment joining into a continuous lagging strand. To date, the models of enzymatic machinery that removes the RNA-DNA primers have come almost exclusively from biochemical reconstitution studies and some genetic interaction assays, and there is little direct evidence to confirm these models. One obstacle to elucidating Okazaki fragment processing has been the lack of methods that can directly examine primer removal in vivo. In this study, we developed an electron microscopy assay that can visualize nucleotide flap structures on DNA replication forks in fission yeast (Schizosaccharomyces pombe). With this assay, we first demonstrated the generation of flap structures during Okazaki fragment processing in vivo. The mean and median lengths of the flaps in wild-type cells were ϳ51 and ϳ41 nucleotides, respectively. We also used yeast mutants to investigate the impact of deleting key DNA replication nucleases on these flap structures. Our results provided direct in vivo evidence for a previously proposed flap cleavage pathway and the critical function of Dna2 and Fen1 in cleaving these flaps. In addition, we found evidence for another previously proposed exonucleolytic pathway involving RNA-DNA primer digestion by exonucleases RNase H2 and Exo1. Taken together, our observations suggest a dual mechanism for Okazaki fragment maturation in lagging strand synthesis and establish a new strategy for interrogation of this fascinating process.In eukaryotic cells, DNA pol 2 ⑀ and pol ␦ direct the synthesis of leading and lagging strand DNA, respectively (1, 2). The antiparallel nature of DNA and the unique 5Ј to 3Ј direction of DNA synthesis by all DNA polymerases make the synthesis of leading strand continuous and lagging strand discontinuous. In the lagging strand, Okazaki fragments are synthesized first (3). The average size of Okazaki fragments in eukaryotic cells is ϳ150 -200 nucleotides (nt) (4). Because DNA polymerases lack de novo DNA synthesis activity, each Okazaki fragment contains an RNA-DNA primer at its 5Ј-end, and this primer is synthesized with low fidelity by primase-DNA pol ␣ complex (5-7). DNA ligase I is responsible for joining Okazaki fragments together to form a continuous lagging strand. Because DNA ligase I is unable to join DNA to RNA, the RNA-DNA primers must be removed from each Okazaki fragment to complete lagging strand DNA synthesis and maintain genomic stability.The mechanism underlying the removal of RNA-DNA primers from Okazaki fragments remains uncertain. Over the previous 20 years, three models have been proposed to explain how these primers are removed (8). In the first model, the RNA-DNA primers are hydrolyzed directly by RNase H2 and DNA exonucleases, such as Fen1 (the exonu...

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confidence: 99%

Direct Visualization of RNA-DNA Primer Removal from Okazaki Fragments Provides Support for Flap Cleavage and Exonucleolytic Pathways in Eukaryotic Cells

Liu

Wang

et al. 2017

Journal of Biological Chemistry

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“…Ligation of the Okazaki fragment to the adjacent DNA then extends the lagging strand. The activity of the archaeal flap endonuclease (Fen1, encoded by TK1281 in Thermococcus kodakarensis) has been well documented (18)(19)(20)(21), but the 5=-to-3= exonuclease activity of GAN (10) and the activity of an RNase HII (14,(22)(23)(24) could also provide alternative mechanisms for the removal of archaeal primer sequences.…”

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The GAN Exonuclease or the Flap Endonuclease Fen1 and RNase HII Are Necessary for Viability of Thermococcus kodakarensis

et al. 2017

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Many aspects of and factors required for DNA replication are conserved across all three domains of life, but there are some significant differences surrounding lagging-strand synthesis. In Archaea, a 5=-to-3= exonuclease, related to both bacterial RecJ and eukaryotic Cdc45, that associates with the replisome specifically through interactions with GINS was identified and designated GAN (for GINS-associated nuclease). Despite the presence of a well-characterized flap endonuclease (Fen1), it was hypothesized that GAN might participate in primer removal during Okazaki fragment maturation, and as a Cdc45 homologue, GAN might also be a structural component of an archaeal CMG (Cdc45, MCM, and GINS) replication complex. We demonstrate here that, individually, either Fen1 or GAN can be deleted, with no discernible effects on viability and growth. However, deletion of both Fen1 and GAN was not possible, consistent with both enzymes catalyzing the same step in primer removal from Okazaki fragments in vivo. RNase HII has also been proposed to participate in primer processing during Okazaki fragment maturation. Strains with both Fen1 and RNase HII deleted grew well. GAN activity is therefore sufficient for viability in the absence of both RNase HII and Fen1, but it was not possible to construct a strain with both RNase HII and GAN deleted. Fen1 alone is therefore insufficient for viability in the absence of both RNase HII and GAN. The ability to delete GAN demonstrates that GAN is not required for the activation or stability of the archaeal MCM replicative helicase. IMPORTANCEThe mechanisms used to remove primer sequences from Okazaki fragments during lagging-strand DNA replication differ in the biological domains. Bacteria use the exonuclease activity of DNA polymerase I, whereas eukaryotes and archaea encode a flap endonuclease (Fen1) that cleaves displaced primer sequences. RNase HII and the GINS-associated exonuclease GAN have also been hypothesized to assist in primer removal in Archaea. Here we demonstrate that in Thermococcus kodakarensis, either Fen1 or GAN activity is sufficient for viability. Furthermore, GAN can support growth in the absence of both Fen1 and RNase HII, but Fen1 and RNase HII are required for viability in the absence of GAN.KEYWORDS Archaea, CMG complex, DNA replication, GINS-associated nuclease GAN, Okazaki fragment, RNase HII, flap endonuclease Fen1 D NA replication is catalyzed by a protein complex, designated the replisome, with structural and catalytic components tightly regulated, intertwined, and coordinated to facilitate both leading-and lagging-strand synthesis. The replisome composition is

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“…Many 5Ј-3Ј-exonucleases catalyze the removal of a ssDNA 5Ј-tail on duplex DNA arising from a nick in the DNA (20,21). These ssDNA tails are often referred to as "flaps" or "overhangs," terms that we will use interchangeably.…”

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“…4A (38, 39). The presence of a duplex opposite to a 5Ј flap strand enhances the efficiency of cleavage by FEN1 (21). In order to examine the effect of a duplex opposite to the flap on gp6 flap endonuclease activity, it is necessary to circumvent the potent 5Ј-exonuclease activity that would rapidly remove this annealed oligonucleotide used to A, the efficiency of flap endonuclease activity of gp6 on substrate having a 2-nt (A2:B1) or 6-nt (A3:B1) 5Ј-overhang was examined using the DNA substrates shown in the inset.…”

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Flap Endonuclease Activity of Gene 6 Exonuclease of Bacteriophage T7

Mitsunobu

Zhu

Lee

et al. 2014

Journal of Biological Chemistry

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Background: Flap endonucleases remove 5Ј-single-stranded DNA termini. Results: T7 gene 6 exonuclease is a flap endonuclease that cleaves 5Ј-single-stranded termini one nucleotide into the duplex. Conclusion:The flap endonuclease activity catalyzes the removal 5Ј-single-stranded tails arising from duplex DNA. Significance: The specificity of gene 6 protein identifies it as a flap endonuclease that can remove unusual structures of recombination and replication.

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The Wonders of Flap Endonucleases: Structure, Function, Mechanism and Regulation

Cited by 49 publications

References 102 publications

Direct Visualization of RNA-DNA Primer Removal from Okazaki Fragments Provides Support for Flap Cleavage and Exonucleolytic Pathways in Eukaryotic Cells

Direct Visualization of RNA-DNA Primer Removal from Okazaki Fragments Provides Support for Flap Cleavage and Exonucleolytic Pathways in Eukaryotic Cells

The GAN Exonuclease or the Flap Endonuclease Fen1 and RNase HII Are Necessary for Viability of Thermococcus kodakarensis

Flap Endonuclease Activity of Gene 6 Exonuclease of Bacteriophage T7

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