Related Experiment Video

Updated: May 21, 2026

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity
09:45

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Published on: January 29, 2018

Adenovirus regulates sumoylation of Mre11-Rad50-Nbs1 components through a paralog-specific mechanism

Sook-Young Sohn1, Patrick Hearing

  • 1Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, New York, USA.

Journal of Virology
|June 29, 2012
PubMed

Insights

Adenovirus E4-ORF3 protein induces SUMOylation of the DNA repair Mre11-Rad50-Nbs1 (MRN) complex. This modification is transient and regulated by viral proteins, influencing the DNA damage response.

Area of Science:

  • Molecular Biology
  • Virology
  • Cellular Biology

Background:

  • The Mre11-Rad50-Nbs1 (MRN) complex is crucial for DNA damage response.
  • Adenovirus (Ad) employs viral proteins to subvert MRN function during infection.
  • Ad uses E1B-55K and E4-ORF6 for MRN degradation and E4-ORF3 for nuclear relocalization.

Purpose of the Study:

  • To investigate the post-translational modifications of the MRN complex during Ad5 infection.
  • To determine the role of Ad5 E4-ORF3 protein in MRN modification.
  • To elucidate the regulation of SUMOylation and desumoylation of MRN components.

Main Methods:

  • Western blotting to detect SUMO-1 and SUMO-2 conjugation.
  • Immunofluorescence microscopy to visualize MRN relocalization.
  • Analysis of MRN modification in cells infected with wild-type and mutant Ad5 viruses.

Main Results:

  • Mre11 is modified by SUMO-2, and Nbs1 by both SUMO-1 and SUMO-2 during Ad5 infection.
  • Ad5 E4-ORF3 protein is necessary and sufficient to induce MRN sumoylation.
  • SUMOylation is dependent on MRN relocalization into E4-ORF3 nuclear tracks.
  • SUMO-1 conjugation to Nbs1 is stable in E1B-55K or E4-ORF6 mutant infections, suggesting regulated desumoylation.
  • Viral DNA replication inhibition blocks SUMO-2 deconjugation, indicating a late-phase desumoylation process.

Conclusions:

  • Ad5 E4-ORF3 protein induces SUMO-1 and SUMO-2 conjugation to Nbs1 and Mre11, respectively.
  • Paralog-specific and transient SUMOylation/desumoylation of MRN is regulated by Ad5.
  • E4-ORF3-mediated MRN relocalization influences the cellular DNA damage response through SUMOylation.

More Related Videos

Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells10:22

Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells

Published on: November 12, 2015

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells09:40

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

Published on: November 2, 2017

Related Concept Videos

Covalently Linked Protein Regulators02:04

Covalently Linked Protein Regulators

Proteins can undergo many types of post-translational modifications, often in response to changes in their environment. These modifications play an important role in the function and stability of these proteins. Covalently linked molecules include functional groups, such as methyl, acetyl, and phosphate groups, and also small proteins, such as ubiquitin. There are around 200 different types of covalent regulators that have been identified.
These groups modify specific amino acids in a protein.
Leaky Scanning02:28

Leaky Scanning

During most eukaryotic translation processes, the small 40S ribosome subunit scans an mRNA from its 5' end until it encounters the first start AUG codon. The large 60S ribosomal subunit then joins the smaller one to initiate protein synthesis. The location of the translation initiation is largely determined by the nucleotides near the start codon as there may be multiple translation initiation sites present on the mRNA.  Marilyn Kozak discovered that the sequence RCCAUGG (where R stands for...
Regulation of the Unfolded Protein Response01:31

Regulation of the Unfolded Protein Response

Inositol-requiring kinase one or IRE1 is the most conserved eukaryotic unfolded protein response (UPR) receptor. It is a type I transmembrane protein kinase receptor with a distinctive site-specific RNase activity. As the binding mechanics of the misfolded proteins with the N-terminal domain of IRE-1 are unclear, three binding models — direct, indirect, and allosteric -- are proposed for receptor activation. Nevertheless, it is known that once a misfolded protein associates with IRE1, it...
Conservative Site-specific Recombination and Phase Variation02:53

Conservative Site-specific Recombination and Phase Variation

Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
Protein Complex Assembly02:41

Protein Complex Assembly

Proteins can form homomeric complexes with another unit of the same protein or heteromeric complexes with different types.  Most protein complexes self-assemble spontaneously via ordered pathways, while some proteins need assembly factors that guide their proper assembly. Despite the crowded intracellular environment, proteins usually interact with their correct partners and form functional complexes.
Many viruses self-assemble into a fully functional unit using the infected host cell to...
Regulation of Nuclear Protein Sorting01:45

Regulation of Nuclear Protein Sorting

Nuclear protein sorting regulates nucleus composition and gene expression, crucial for determining the fate of a eukaryotic cell. Hence, the entry and exit of molecules across the nuclear envelope is a tightly controlled process. Nuclear protein sorting can be inhibited by one of the following ways: 1) masking cargo signal sequences, 2) modifying the nuclear receptor's affinity for cargo, 3) controlling the nuclear pore size, 4) retaining the cargo during its transit to the cytosol or the...