The conserved heterodimeric endonuclease Mus81–Eme1/Mms4 plays an important role in the maintenance of genomic integrity in eukaryotic cells. Here, we show that budding yeast Mus81–Mms4 is strictly regulated during the mitotic cell cycle by Cdc28 (CDK)- and Cdc5 (Polo-like kinase)-dependent phosphorylation of the non-catalytic subunit Mms4. The phosphorylation of this protein occurs only after bulk DNA synthesis and before chromosome segregation, and is absolutely necessary for the function of the Mus81–Mms4 complex. Consistently, a phosphorylation-defective mms4 mutant shows highly reduced nuclease activity and increases the sensitivity of cells lacking the RecQ-helicase Sgs1 to various agents that cause DNA damage or replicative stress. The mode of regulation of Mus81–Mms4 restricts its activity to a short period of the cell cycle, thus preventing its function during chromosome replication and the negative consequences for genome stability derived from its nucleolytic action. Yet, the controlled Mus81–Mms4 activity provides a safeguard mechanism to resolve DNA intermediates that may remain after replication and require processing before mitosis.

The infectious bursal disease virus T=13 viral particle is composed of two major proteins, VP2 and VP3. Here, we show that the molecular basis of the conformational flexibility of the major capsid protein precursor, pVP2, is an amphipatic alpha helix formed by the sequence GFKDIIRAIR. VP2 containing this alpha helix is able to assemble into the T=13 capsid only when expressed as a chimeric protein with an N-terminal His tag. An amphiphilic alpha helix, which acts as a conformational switch, is thus responsible for the inherent structural polymorphism of VP2. The His tag mimics the VP3 C-terminal region closely and acts as a molecular triggering factor. Using cryo-electron microscopy difference imaging, both polypeptide elements were detected on the capsid inner surface. We propose that electrostatic interactions between these two morphogenic elements are transmitted to VP2 to acquire the competent conformations for capsid assembly.

Genome-binding proteins with scaffolding and/or regulatory functions are common in living organisms and include histones in eukaryotic cells, histone-like proteins in some double-stranded DNA (dsDNA) viruses, and the nucleocapsid proteins of single-stranded RNA viruses. dsRNA viruses nevertheless lack these ribonucleoprotein (RNP) complexes and are characterized by sharing an icosahedral T=2 core involved in the metabolism and insulation of the dsRNA genome. The birnaviruses, with a bipartite dsRNA genome, constitute a well-established exception and have a single-shelled T=13 capsid only. Moreover, as in many negative single-stranded RNA viruses, the genomic dsRNA is bound to a nucleocapsid protein (VP3) and the RNA-dependent RNA polymerase (VPg). We used electron microscopy and functional analysis to characterize these RNP complexes of infectious bursal disease virus, the best characterized member of the Birnaviridae family. Mild disruption of viral particles revealed that VP3, the most abundant core protein, present at approximately 450 copies per virion, is found in filamentous material tightly associated with the dsRNA. We developed a method to purify RNP and VPg-dsRNA complexes. Analysis of these complexes showed that they are linear molecules containing a constant amount of protein. Sensitivity assays to nucleases indicated that VP3 renders the genomic dsRNA less accessible for RNase III without introducing genome compaction. Additionally, we found that these RNP complexes are functionally competent for RNA synthesis in a capsid-independent manner, in contrast to most dsRNA viruses.

Mgs1, the budding yeast homolog of mammalian Werner helicase-interacting protein 1 (WRNIP1/WHIP), contributes to genome stability during undisturbed replication and in response to DNA damage. A ubiquitin-binding zinc finger (UBZ) domain directs human WRNIP1 to nuclear foci, but the functional significance of its presence and the relevant ubiquitylation targets that this domain recognizes have remained unknown. Here, we provide a mechanistic basis for the ubiquitin-binding properties of the protein. We show that in yeast an analogous domain exclusively mediates the damage-related activities of Mgs1. By means of preferential physical interactions with the ubiquitylated forms of the replicative sliding clamp, proliferating cell nuclear antigen (PCNA), the UBZ domain facilitates recruitment of Mgs1 to sites of replication stress. Mgs1 appears to interfere with the function of polymerase δ, consistent with our observation that Mgs1 inhibits the interaction between the polymerase and PCNA. Our identification of Mgs1 as a UBZ-dependent downstream effector of ubiquitylated PCNA suggests an explanation for the ambivalent role of the protein in damage processing.

The RAD6/RAD18 pathway of DNA damage tolerance overcomes unrepaired lesions that block replication forks. It is subdivided into two branches: translesion DNA synthesis, which is frequently error prone, and the error-free DNA-damage-avoidance subpathway. Here, we show that Rad5(HLTF/SHPRH), which mediates the error-free branch, has a major role in the response to DNA damage caused by methyl methanesulfonate (MMS) during chromosome replication, whereas translesion synthesis polymerases make only a minor contribution. Both the ubiquitin-ligase and the ATPase/helicase activities of Rad5 are necessary for this cellular response. We show that Rad5 is required for the progression of replication forks through MMS-damaged DNA. Moreover, supporting its role during replication, this protein reaches maximum levels during S phase and forms subnuclear foci when replication occurs in the presence of DNA damage. Thus, Rad5 ensures the completion of chromosome replication under DNA-damaging conditions while minimizing the risk of mutagenesis, thereby contributing significantly to genome integrity maintenance.

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a double-stranded RNA virus that causes a highly contagious disease in young chickens leading to significant economic losses in the poultry industry. The VP2 protein, the only structural component of the IBDV icosahedral capsid, spontaneously assembles into T‫1؍‬ subviral particles (SVP) when individually expressed as a chimeric gene. We have determined the crystal structure of the T‫1؍‬ SVP to 2.60 Å resolution. Our results show that the 20 trimeric VP2 clusters forming the T‫1؍‬ shell are further stabilized by calcium ions located at the threefold icosahedral axes. The structure also reveals a new unexpected domain swapping that mediates interactions between adjacent trimers: a short helical segment located close to the end of the long C-terminal arm of VP2 is projected toward the threefold axis of a neighboring VP2 trimer, leading to a complex network of interactions that increases the stability of the T‫1؍‬ particles. Analysis of crystal packing shows that the exposed capsid residues, His253 and Thr284, determinants of IBDV virulence and the adaptation of the virus to grow in cell culture, are involved in particle-particle interactions.Infectious bursal disease virus (IBDV), the best characterized member of the Birnaviridae family, is an important avian pathogen that inflicts major economic losses to the poultry industry (42). Infection with virulent IBDV strains causes the destruction of pre-B-lymphocyte populations contained within the bursa of Fabricius and, as a consequence, the immunosuppression of affected birds (4).IBDV possesses a bipartite double-stranded RNA genome encoding five mature polypeptides. The major RNA segment contains two partially overlapping open reading frames (ORFs): the first one codes for the 16-kDa polypeptide VP5 that appears to play an important role in virus dissemination and virulence (28, 44) and the second one codes for the virus polyprotein (110 kDa). The smaller RNA segment contains a single ORF encoding protein VP1, which is the virus RNAdependent RNA polymerase (11).The virus polyprotein is cotranslationally processed to give three polypeptides, named pVP2 (54 kDa), VP4 (27 kDa), and VP3 (29 kDa). VP4 is the viral protease responsible for the polyprotein cleavage (23). pVP2 (the VP2 precursor) interacts with VP3, initiating the capsid assembly pathway in which VP3 plays an essential scaffolding role (30). pVP2 is further cleaved at its C-terminal end by an unknown mechanism releasing the mature VP2 (47 kDa). This second processing event requires capsid assembly (6).The IBDV capsid consists of a single shell formed by 260 trimers of protein VP2 organized in a Tϭ13 icosahedral lattice (2, 5). Previous work aimed at the characterization of the IBDV assembly process revealed that independent expression of the VP2 coding region from chimeric genes leads to the assembly of icosahedral Tϭ1 subviral particles (SVPs) of ϳ23 nm in diameter, whereas pVP2 expression results in the formation of tubular s...

The structure-specific Mus81-Eme1/Mms4 endonuclease contributes importantly to DNA repair and genome integrity maintenance. Here, using budding yeast, we have studied its function and regulation during the cellular response to DNA damage and show that this endonuclease is necessary for successful chromosome replication and cell survival in the presence of DNA lesions that interfere with replication fork progression. On the contrary, Mus81-Mms4 is not required for coping with replicative stress originated by acute treatment with hydroxyurea (HU), which causes fork stalling. Despite its requirement for dealing with DNA lesions that hinder DNA replication, Mus81-Mms4 activation is not induced by DNA damage at replication forks. Full Mus81-Mms4 activity is only acquired when cells finish S-phase and the endonuclease executes its function after the bulk of genome replication is completed. This post-replicative mode of action of Mus81-Mms4 limits its nucleolytic activity during S-phase, thus avoiding the potential cleavage of DNA substrates that could cause genomic instability during DNA replication. At the same time, it constitutes an efficient fail-safe mechanism for processing DNA intermediates that cannot be resolved by other proteins and persist after bulk DNA synthesis, which guarantees the completion of DNA repair and faithful chromosome replication when the DNA is damaged.

Infectious bursal disease virus (IBDV), a double-stranded RNA (dsRNA) virus belonging to the Birnaviridae family, is an economically important avian pathogen. The IBDV capsid is based on a single-shelled T‫31؍‬ lattice, and the only structural subunits are VP2 trimers. During capsid assembly, VP2 is synthesized as a protein precursor, called pVP2, whose 71-residue C-terminal end is proteolytically processed. The conformational flexibility of pVP2 is due to an amphipathic ␣-helix located at its C-terminal end. VP3, the other IBDV major structural protein that accomplishes numerous roles during the viral cycle, acts as a scaffolding protein required for assembly control. Here we address the molecular mechanism that defines the multimeric state of the capsid protein as hexamers or pentamers. We used a combination of three-dimensional cryo-electron microscopy maps at or close to subnanometer resolution with atomic models. Our studies suggest that the key polypeptide element, the C-terminal amphipathic ␣-helix, which acts as a transient conformational switch, is bound to the flexible VP2 C-terminal end. In addition, capsid protein oligomerization is also controlled by the progressive trimming of its C-terminal domain. The coordination of these molecular events correlates viral capsid assembly with different conformations of the amphipathic ␣-helix in the precursor capsid, as a five-␣-helix bundle at the pentamers or an open star-like conformation at the hexamers. These results, reminiscent of the assembly pathway of positive single-stranded RNA viruses, such as nodavirus and tetravirus, add new insights into the evolutionary relationships of dsRNA viruses.Infectious bursal disease virus (IBDV) infects young chickens, causing a highly contagious disease that targets the precursors of antibody-producing B cells (4). This avian disease is economically relevant to the poultry industry worldwide (45). IBDV belongs to the genus Avibirnavirus in the family Birnaviridae. The IBDV virion is spherical, with a diameter of ϳ650 to 700 Å, and contains two double-stranded RNA (dsRNA) segments, of 3.2 and 2.8 kbp (segments A and B, respectively), and five encoded mature proteins (VP1 to VP5) (15). Most of these proteins are encoded by segment A, which has two partially overlapping open reading frames; the first one encodes a dispensable nonstructural protein, VP5 (17 kDa), and the second encodes three proteins synthesized as a polyprotein (110 kDa). Segment B has a single open reading frame encoding VP1 (98 kDa), the RNA-dependent RNA polymerase (47). The viral polyprotein is cotranslationally processed by the viral protease VP4 (5, 17), rendering the VP2 precursor, termed pVP2 (512 residues; 54.4 kDa), VP3 (29 kDa), and VP4 (27 kDa). Most of the pVP2 C-terminal region is further processed at three Ala-Ala bonds, which are secondary VP4 targets (positions 487, 494, and 501) (38). The resulting intermediate pVP2 is again cleaved, by an unknown mechanism, between residues 441 and 442, giving the mature VP2 polypeptide (47 kDa) (Fig. ...