RNAi-Based Suppressor Screens Reveal Genetic Interactions Between the CRL2LRR-1 E3-Ligase and the DNA Replication Machinery in Caenorhabditis elegans

In eukaryotic cells, DNA replication licensing is precisely regulated to ensure that the initiation of genomic DNA replication in S phase occurs once and only once for each mitotic cell division. A key regulatory mechanism by which DNA re-replication is suppressed is the S phase-dependent proteolysis of Cdt1, an essential replication protein for licensing DNA replication origins by loading the Mcm2-7 replication helicase for DNA duplication in S phase. Cdt1 degradation is mediated by CRL4Cdt2 ubiquitin E3 ligase, which further requires Cdt1 binding to proliferating cell nuclear antigen (PCNA) through a PIP box domain in Cdt1 during DNA synthesis. Recent studies found that Cdt2, the specific subunit of CRL4Cdt2 ubiquitin E3 ligase that targets Cdt1 for degradation, also contains an evolutionarily conserved PIP box-like domain that mediates the interaction with PCNA. These findings suggest that the initiation and elongation of DNA replication or DNA damage-induced repair synthesis provide a novel mechanism by which Cdt1 and CRL4Cdt2 are both recruited onto the trimeric PCNA clamp encircling the replicating DNA strands to promote the interaction between Cdt1 and CRL4Cdt2. The proximity of PCNA-bound Cdt1 to CRL4Cdt2 facilitates the destruction of Cdt1 in response to DNA damage or after DNA replication initiation to prevent DNA re-replication in the cell cycle. CRL4Cdt2 ubiquitin E3 ligase may also regulate the degradation of other PIP box-containing proteins, such as CDK inhibitor p21 and histone methylase Set8, to regulate DNA replication licensing, cell cycle progression, DNA repair, and genome stability by directly interacting with PCNA during DNA replication and repair synthesis.

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DNA replication machinery: Insights from in vitro single-molecule approaches

Computational and Structural Biotechnology Journal ◽

10.1016/j.csbj.2021.04.013 ◽

2021 ◽

Vol 19 ◽

pp. 2057-2069

Author(s):

Rebeca Bocanegra ◽

G.A. Ismael Plaza ◽

Carlos R. Pulido ◽

Borja Ibarra

Keyword(s):

Dna Replication ◽

Single Molecule ◽

Replication Machinery

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The replication machinery of LUCA: common origin of DNA replication and transcription

BMC Biology ◽

10.1186/s12915-020-00800-9 ◽

2020 ◽

Vol 18 (1) ◽

Cited By ~ 6

Author(s):

Eugene V. Koonin ◽

Mart Krupovic ◽

Sonoko Ishino ◽

Yoshizumi Ishino

Keyword(s):

Dna Replication ◽

Common Origin ◽

Replication Machinery ◽

Origin Of Dna Replication

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Regulation of Caenorhabditis elegans lifespan by a proteasomal E3 ligase complex

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0700638104 ◽

2007 ◽

Vol 104 (14) ◽

pp. 5947-5952 ◽

Cited By ~ 90

Author(s):

A. Ghazi ◽

S. Henis-Korenblit ◽

C. Kenyon

Keyword(s):

Caenorhabditis Elegans ◽

E3 Ligase

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Interacting genes required for pharyngeal excitation by motor neuron MC in Caenorhabditis elegans.

Genetics ◽

10.1093/genetics/141.4.1365 ◽

1995 ◽

Vol 141 (4) ◽

pp. 1365-1382 ◽

Cited By ~ 11

Author(s):

D M Raizen ◽

R Y Lee ◽

L Avery

Keyword(s):

Caenorhabditis Elegans ◽

Genetic Interactions ◽

Neuron Type ◽

Loss Of Function ◽

Ach Release ◽

Pharyngeal Muscle ◽

Recessive Mutations ◽

Allele Specific ◽

Pharyngeal Pumping ◽

Pumping Rates

Abstract We studied the control of pharyngeal excitation in Caenorhabditis elegans. By laser ablating subsets of the pharyngeal nervous system, we found that the MC neuron type is necessary and probably sufficient for rapid pharyngeal pumping. Electropharyngeograms showed that MC transmits excitatory postsynaptic potentials, suggesting that MC acts as a neurogenic pacemaker for pharyngeal pumping. Mutations in genes required for acetylcholine (ACh) release and an antagonist of the nicotinic ACh receptor (nAChR) reduced pumping rates, suggesting that a nAChR is required for MC transmission. To identify genes required for MC neurotransmission, we screened for mutations that cause slow pumping but no other defects. Mutations in two genes, eat-2 and eat-18, eliminated MC neurotransmission. A gain-of-function eat-18 mutation, ad820sd, and a putative loss-of-function eat-18 mutation, ad1110, both reduced the excitation of pharyngeal muscle in response to the nAChR agonists nicotine and carbachol, suggesting that eat-18 is required for the function of a pharyngeal nAChR. Fourteen recessive mutations in eat-2 fell into five complementation classes. We found allele-specific genetic interactions between eat-2 and eat-18 that correlated with complementation classes of eat-2. We propose that eat-18 and eat-2 function in a multisubunit protein complex involved in the function of a pharyngeal nAChR.

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The TREX2 3′→ 5′ Exonuclease Physically Interacts with DNA Polymerase δ and Increases Its Accuracy

The Scientific World JOURNAL ◽

10.1100/tsw.2002.99 ◽

2002 ◽

Vol 2 ◽

pp. 275-281 ◽

Cited By ~ 15

Author(s):

Igor V. Shevelev ◽

Kristijan Ramadan ◽

Ulrich Hubscher

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Error Rates ◽

High Fidelity ◽

Replication Machinery ◽

Dna Polymerase Δ ◽

Pol I ◽

Calf Thymus ◽

Mutation Assay ◽

Forward Mutation

Proofreading function by the 3′→ 5′ exonuclease of DNA polymerase δ (pol δ) is consistent with the observation that deficiency of the associated exonuclease can lead to a strong mutation phenotype, high error rates during DNA replication, and ultimately cancer. We have isolated pol δdfrom isotonic (pol δi) and detergent (pol δd) calf thymus extracts. Pol δdhad a 20-fold higher ratio of exonuclease to DNA polymerase than pol δi. This was due to the physical association of the TREX2 exonuclease to pol δd, which was missing from pol δi. Pol δdwas fivefold more accurate than pol δiunder error-prone conditions (1 μM dGTP and 20 dATP, dCTP, and dTTP) in a M13mp2 DNA forward mutation assay, and fourfold more accurate in an M13mp2T90 reversion assay. Under error-free conditions (20 μM each of the four dNTPs), however, both polymerases showed equal fidelity. Our data suggested that autonomous 3′→ 5′ exonucleases, such as TREX2, through its association with pol I can guarantee high fidelity under difficult conditions in the cell (e.g., imbalance of dNTPs) and can add to the accuracy of the DNA replication machinery, thus preventing mutagenesis.

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