ECR1

ECR1 (essential for chromosome replication 1) is a 52-kDa putative E3 ubiquitin ligase, bearing divergent RING and TRAF-Sina-like zinc-binding domains, that couples the ubiquitin-proteasome system to controlled chromosome replication during Toxoplasma gondii tachyzoite division (PMID:28830940). It is dynamically staged in the cell cycle, appearing first in the centrocone (the spindle compartment of the nuclear envelope) in early S phase, then relocating to the nucleus at peak expression in late S phase before being downregulated after nuclear division (PMID:28830940). ECR1 associates with the ubiquitin-mediated degradation machinery and the minichromosome maintenance (MCM) complex, and its loss stabilizes MCM2, indicating that ECR1 promotes MCM2 turnover to restrain DNA replication (PMID:28830940). It also forms a stable complex with the CDK-related kinase TgCrk5, with which it shares expression timing and centrocone co-localization, linking ECR1 to cell-cycle kinase signaling (PMID:28830940). Loss of ECR1 function causes lethal, uncontrolled chromosome replication resembling a topoisomerase-defective phenotype, establishing its essential role in gating replication and mitotic progression (PMID:28830940).

1. 2017 Medium

   Identifying ECR1 as the gene complementing a temperature-sensitive replication mutant established a previously uncharacterized RING/TRAF-Sina-domain protein as an essential, cell-cycle-staged regulator of tachyzoite division.

   Evidence Forward genetic complementation of a ts mutant with domain analysis and cell-cycle-staged live/fixed imaging in T. gondii

   PMID:28830940

   Open questions at the time

   • E3 ligase catalytic activity not demonstrated biochemically
   • The signal driving centrocone-to-nucleus relocalization is unknown
2. 2017 Medium

   Phenotyping ECR1 loss at restrictive temperature showed it is required to gate chromosome replication, since its absence produces lethal uncontrolled replication like a topoisomerase-defective mutant.

   Evidence Temperature-sensitive mutant complementation and phenotypic analysis at 40°C in T. gondii

   PMID:28830940

   Open questions at the time

   • Whether the replication defect is a direct consequence of lost ligase activity is untested
   • Downstream effects on mitotic progression not resolved mechanistically
3. 2017 Medium

   Proteomic mapping placed ECR1 in physical contact with the ubiquitin-degradation machinery and the MCM complex, and stabilization of MCM2 upon ECR1 loss provided a candidate substrate linking ECR1 to replication control.

   Evidence Co-IP mass spectrometry and MCM2 protein stability assay upon ECR1 loss in T. gondii

   PMID:28830940

   Open questions at the time

   • Direct ubiquitination of MCM2 by ECR1 not demonstrated in vitro
   • Whether MCM2 degradation is the sole or primary effector of the replication phenotype is unresolved
4. 2017 Medium

   Co-IP and co-localization identified a stable ECR1–TgCrk5 complex, connecting the putative ligase to a CDK-related kinase acting at the centrocone.

   Evidence Co-immunoprecipitation and co-localization imaging in T. gondii

   PMID:28830940

   Open questions at the time

   • Whether TgCrk5 phosphorylates ECR1 or regulates its activity is unknown
   • The functional consequence of the complex for replication control is uncharacterized

• Whether ECR1 possesses demonstrable E3 ligase activity and directly ubiquitinates MCM2, and how the ECR1–TgCrk5 complex is coordinated to gate replication, remain open.
• No in vitro reconstitution of ECR1 ubiquitin ligase activity
• No structural model of the RING/TRAF-Sina domains
• Mechanism linking TgCrk5 kinase to ECR1-mediated MCM2 turnover unknown