Atomic structure of an archaeal GAN suggests its dual roles as an exonuclease in DNA repair and a CMG component in DNA replication

Oyama, Takuji; Ishino, Sonoko; Shirai, Tomoyuki; Yamagami, Takeshi; Nagata, Mariko; Ogino, Hiromi; Kusunoki, Masami; Ishino, Yuji

doi:10.1093/nar/gkw789

Cited by 24 publications

(63 citation statements)

References 39 publications

(56 reference statements)

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“…The proposal that GAN may also function as a structural homologue of Cdc45 as a component of an archaeal CMG-like complex is consistent with the established stimulatory interaction of GAN and GINS (10,12), but the ability to delete GAN eliminates that this is an essential role for GAN in archaeal DNA replication. It is plausible that Fen1 can replace GAN in such a complex, but more likely, stimulation of the archaeal replicative helicase (MCM) by GINS alone is sufficient for robust helicase activity in vivo.…”

Section: Figsupporting

confidence: 56%

“…Based on homology with RecJ and Cdc45, it was proposed that GAN might have two functions: the exonucleolytic removal of primer sequences during Okazaki fragment maturation and stimulation of replication as a structural component of a CMG-like complex (10)(11)(12). The construction of strains with GAN deleted eliminated both roles as essential, and to address the requirement for GAN exonuclease activity, the two aspartate residues known to be essential for catalysis were replaced with alanines (10, 12).…”

Section: Resultsmentioning

confidence: 99%

“…4). The biochemical properties of Fen1 are consistent with the endonucleolytic cleavage of displaced RNA primers, but purified GAN has DNA-specific exonucleolytic activity, apparently inconsistent with the processing of RNA flaps (10,12). However, archaeal DNA primases can use deoxynucleoside triphosphates (dNTPs) to generate DNA-based primers (31), raising the possibility that, in vivo, GAN removes DNA primers and/or that GAN gains RNase activity in vivo when associated with the replisome.…”

Section: Discussionmentioning

confidence: 98%

“…Given that Fen1 is not essential for T. kodakarensis, there must be an alternative mechanism(s) to remove the primers, and here we have investigated whether the exonuclease activity of GAN (10,12) and/or the degrading activity of RNase HII within an RNA-DNA hybrid provides alternative mechanisms (14,(22)(23)(24).…”

Section: Discussionmentioning

confidence: 99%

“…Although sequence conservation and homology modeling have identified many components of the archaeal replisome, archaeon-specific replication factors have also been isolated from complexes formed with established components of the replication machinery (4)(5)(6)(7)(8)(9)(10). One such factor, encoded by TK1252 in Thermococcus kodakarensis and designated GAN (GINS-associated nuclease), shares sequence and structural similarities with the bacterial RecJ and eukaryotic Cdc45 proteins (11,12). Purified GAN has 5=-to-3= exonuclease activity, stimulated by association with GINS and, based on Cdc45 homology, was suggested to participate in archaeal replication as a structural component of a complex modeled on a eukaryotic helicase, the CMG (Cdc45, MCM, and GINS) complex (10).…”

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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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Section: Figsupporting

confidence: 56%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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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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The small subunit of DNA polymerase D (DP1) associates with GINS–GAN complex of the thermophilic archaea in Thermococcus sp. 4557

Chen

et al. 2019

MicrobiologyOpen

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The eukaryotic GINS, Cdc45, and minichromosome maintenance proteins form an essential complex that moves with the DNA replication fork. The GINS protein complex has also been reported to associate with DNA polymerase. In archaea, the third domain of life, DNA polymerase D (PolD) is essential for DNA replication, and the genes encoding PolDs exist only in the genomes of archaea. The archaeal GAN (GINS‐associated nuclease) is believed to be a homolog of the eukaryotic Cdc45. In this study, we found that the Thermococcus sp. 4557 DP1 (small subunit of PolD) interacted with GINS15 in vitro, and the 3′–5′ exonuclease activity of DP1 was inhibited by GINS15. We also demonstrated that the GAN, GINS15, and DP1 proteins interact to form a complex adapting a GAN–GINS15–DP1 order. The results of this study imply that the complex constitutes a core of the DNA replisome in archaea.

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Initiation of DNA Replication in the Archaea

Bell

2017

Advances in Experimental Medicine and Biology

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Organisms within the archaeal domain of life possess a simplified version of the eukaryotic DNA replication machinery. While some archaea possess a bacterial-like mode of DNA replication with single origins of replication per chromosome, the majority of species characterized to date possess chromosomes with multiple replication origins. Genetic, structural, and biochemical studies have revealed the nature of archaeal origin specification. Recent work has begun to shed light on the mechanisms of replication initiation in these organisms.

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Atomic structure of an archaeal GAN suggests its dual roles as an exonuclease in DNA repair and a CMG component in DNA replication

Cited by 24 publications

References 39 publications

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

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

The small subunit of DNA polymerase D (DP1) associates with GINS–GAN complex of the thermophilic archaea in Thermococcus sp. 4557

Initiation of DNA Replication in the Archaea

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