Specificity and Function of Archaeal DNA Replication Initiator Proteins

Samson, Rachel Y.; Xu, Yanqun; Gadelha, Catarina; Stone, Todd A.; Faqiri, Jamal N.; Li, Dongfang; Qin, Nan; Pu, Fei; Liang, Yanhui; She, Qunxin; Bell, Stephen D.

doi:10.1016/j.celrep.2013.01.002

Cited by 68 publications

(114 citation statements)

References 44 publications

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“…Although this may simply reflect binding affinity in our in vitro assays conditions, it is conceivable that regulatory modifications to MCM and/or CG components in vivo could affect the strength of interaction we observe. Such a mode of regulation could provide a mechanism for the coordinate control of the three disparate origins present in the Sulfolobus chromosome (27).…”

Section: Discussionmentioning

confidence: 99%

Archaeal orthologs of Cdc45 and GINS form a stable complex that stimulates the helicase activity of MCM

Gristwood²,

Hodgson³

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The regulated recruitment of Cdc45 and GINS is key to activating the eukaryotic MCM(2-7) replicative helicase. We demonstrate that the homohexameric archaeal MCM helicase associates with orthologs of GINS and Cdc45 in vivo and in vitro. Association of these factors with MCM robustly stimulates the MCM helicase activity. In contrast to the situation in eukaryotes, archaeal Cdc45 and GINS form an extremely stable complex before binding MCM. Further, the archaeal GINS • Cdc45 complex contains two copies of Cdc45. Our analyses give insight into the function and evolution of the conserved core of the archaeal/eukaryotic replisome.T he initiation of DNA replication is an important control point in the progression of the cell cycle. In eukaryotes, origins are defined by the initiator protein ORC complex that, via the actions of two additional factors, the helicase coloaders Cdc6 and Cdt1, directs loading of the MCM(2-7) replicative helicase onto doublestranded DNA. Activation of the MCM(2-7) replicative helicase occurs subsequent to recruitment, leading to DNA melting and assembly of the full replisome apparatus (1, 2). Key steps in MCM activation involve a series of phosphorylation-dependent events that promote the sequential association of Cdc45 and the GINS complex with the chromatin-associated MCM double hexamer. These recruitment events are regulated by the CDK and DDK kinases and require the additional accessory factors Sld3/7, Dpb11, and Sld2(3-6). The Cdc45•MCM(2-7)•GINS complex (CMG) forms the core of the eukaryotic replisome, and this 11-subunit assembly appears to be the functional helicase driving fork progression (3-6). The archaeal replication machinery resembles an ancestral form of its eukaryotic counterpart. Archaea possess a simple homohexameric MCM (5, 7). In addition, archaeal homologs of GINS and Cdc45 have been identified (8)(9)(10)(11)(12)(13)(14). In species of the genus Sulfolobus, we have previously demonstrated that the GINS complex interacts with the N-terminal domains of MCM (8). In Sulfolobus, GINS is a dimer of dimers: one subunit, Gins23, is related to the eukaryotic GINS components Psf2 and Psf3, and the second Sulfolobus subunit, Gins15, is related to the eukaryotic Sld5 and Psf1. These sequence relationships have been confirmed by structural studies of the Thermococcus kodakarensis GINS complex that have demonstrated the tetrameric assembly of archaeal (Gins15) 2 • (Gins23) 2 and validated the organizational similarity of the archaeal and eukaryotic GINS complexes (15). Interestingly, Sulfolobus GINS copurifies over the course of eight steps with a further polypeptide that we initially named RecJdbh, based on its observed homology with the presumptive DNA binding domain of the bacterial exonuclease, RecJ (8). Subsequent sequence analyses have revealed a relationship between RecJ and eukaryotic Cdc45, and this has been elegantly confirmed by recent structural studies of eukaryotic Cdc45 (9,11,16,17). We therefore propose renaming RecJdbh as Cdc45. As archaea lack orthologs of Sld2, Sld...

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Section: Discussionmentioning

confidence: 99%

Archaeal orthologs of Cdc45 and GINS form a stable complex that stimulates the helicase activity of MCM

Gristwood²,

Hodgson³

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…These included replication fork-associated proteins such as the single-strand DNA binding protein (SSB); the replicative helicase MCM; the MCM-interacting factor Gins23; sliding clamp subunits PCNA1, 2, and 3; DNA ligase; replicative DNA polymerase polB1; and the clamp loader RFC. In addition, levels of WhiP, a homolog of the eukaryal pre-replicative complex protein Cdt1 that acts as the initiator protein that governs oriC3 (Robinson and Bell, 2007; Samson et al, 2013); the chromatin protein Alba (Bell et al, 2002); the candidate fork regression helicase Hel308; and the recombinase RadA (the RAD51/RecA ortholog) were unaltered in their levels following treatment with HU.…”

Section: Resultsmentioning

confidence: 99%

“…For oriC1 and oriC2 , this can be explained by the marked reduction of the cognate Orc1-1 and Orc1-3 initiator proteins (Robinson et al, 2004; Samson et al, 2013), and the consequent inability to detect Orc1-1 and Orc1-3 at origins by ChIP. However, levels of the WhIP protein, the initiator required for oriC3 , are not altered, and ChIP reveals that this protein remains associated with oriC3 even after treatment with 10 mM HU.…”

Section: Discussionmentioning

confidence: 99%

Hydroxyurea-Mediated Cytotoxicity Without Inhibition of Ribonucleotide Reductase

Liew¹,

Lim

Cohen

et al. 2016

Cell Reports

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SUMMARYIn many organisms, hydroxyurea (HU) inhibits class I ribonucleotide reductase, leading to lowered cellular pools of deoxyribonucleoside triphosphates. The reduced levels for DNA precursors is believed to cause replication fork stalling. Upon treatment of the hyperthermophilic archaeon Sulfolobus solfataricus with HU, we observe dose-dependent cell cycle arrest, accumulation of DNA double-strand breaks, stalled replication forks, and elevated levels of recombination structures. However, Sulfolobus has a HU-insensitive class II ribonucleotide reductase, and we reveal that HU treatment does not significantly impact cellular DNA precursor pools. Profiling of protein and transcript levels reveals modulation of a specific subset of replication initiation and cell division genes. Notably, the selective loss of the regulatory subunit of the primase correlates with cessation of replication initiation and stalling of replication forks. Furthermore, we find evidence for a detoxification response induced by HU treatment.

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“…Studies using chromatin immunoprecipitation (ChIP) followed by analysis of the immunoprecipitated DNA on a wholegenome array chip (ChIP-chip studies) have revealed that the oriC is virtually the only site at which Pyrococcus abyssi Orc1/Cdc6 binds the genome (10). Recent work in Sulfolobus islandicus (11) has shown that two of its three origins are Orc1/Cdc6 dependent. Apart from origin recognition, Orc1/Cdc6 mediates the loading of the replicative helicase (MCM complex) onto DNA (12,13).…”

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confidence: 99%

Characterization of the Replication Initiator Orc1/Cdc6 from the Archaeon Picrophilus torridus

Arora

Goswami

Saha

2013

Journal of Bacteriology

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Eukaryotic DNA replication is preceded by the assembly of prereplication complexes (pre-RCs) at or very near origins in G 1 phase, which licenses origin firing in S phase. The archaeal DNA replication machinery broadly resembles the eukaryal apparatus, though simpler in form. The eukaryotic replication initiator origin recognition complex (ORC), which serially recruits Cdc6 and other pre-RC proteins, comprises six components, Orc1-6. In archaea, a single gene encodes a protein similar to both the eukaryotic Cdc6 and the Orc1 subunit of the eukaryotic ORC, with most archaea possessing one to three Orc1/Cdc6 orthologs. Genome sequence analysis of the extreme acidophile Picrophilus torridus revealed a single Orc1/Cdc6 (PtOrc1/Cdc6). Biochemical analyses show MBP-tagged PtOrc1/Cdc6 to preferentially bind ORB (origin recognition box) sequences. The protein hydrolyzes ATP in a DNA-independent manner, though DNA inhibits MBP-PtOrc1/Cdc6-mediated ATP hydrolysis. PtOrc1/Cdc6 exists in stable complex with PCNA in Picrophilus extracts, and MBP-PtOrc1/Cdc6 interacts directly with PCNA through a PIP box near its C terminus. Furthermore, PCNA stimulates MBP-PtOrc1/Cdc6-mediated ATP hydrolysis in a DNA-dependent manner. This is the first study reporting a direct interaction between Orc1/Cdc6 and PCNA in archaea. The bacterial initiator DnaA is converted from an active to an inactive form by ATP hydrolysis, a process greatly facilitated by the bacterial ortholog of PCNA, the ␤ subunit of Pol III. The stimulation of PtOrc1/Cdc6-mediated ATP hydrolysis by PCNA and the conservation of PCNAinteracting protein motifs in several archaeal PCNAs suggest the possibility of a similar mechanism of regulation existing in archaea. This mechanism may involve other yet to be identified archaeal proteins.

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Specificity and Function of Archaeal DNA Replication Initiator Proteins

Cited by 68 publications

References 44 publications

Archaeal orthologs of Cdc45 and GINS form a stable complex that stimulates the helicase activity of MCM

Archaeal orthologs of Cdc45 and GINS form a stable complex that stimulates the helicase activity of MCM

Hydroxyurea-Mediated Cytotoxicity Without Inhibition of Ribonucleotide Reductase

Characterization of the Replication Initiator Orc1/Cdc6 from the Archaeon Picrophilus torridus

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