RAD18, WRNIP1 and ATMIN promote ATM signalling in response to replication stress.

The DNA replication machinery invariably encounters obstacles that slow replication fork progression, and threaten to prevent complete replication and faithful segregation of sister chromatids. The resulting replication stress activates ATR, the major kinase involved in resolving impaired DNA replication. In addition, replication stress also activates the related kinase ATM, which is required to prevent mitotic segregation errors. However, the molecular mechanism of ATM activation by replication stress is not defined. Here, we show that monoubiquitinated Proliferating Cell Nuclear Antigen (PCNA), a marker of stalled replication forks, interacts with the ATM cofactor ATMIN via WRN-interacting protein 1 (WRNIP1). ATMIN, WRNIP1 and RAD18, the E3 ligase responsible for PCNA monoubiquitination, are specifically required for ATM signalling and 53BP1 focus formation induced by replication stress, not ionising radiation. Thus, WRNIP1 connects PCNA monoubiquitination with ATMIN/ATM to activate ATM signalling in response to replication stress and contribute to the maintenance of genomic stability.

PCNASUMO and Srs2: a model SUMO substrate-effector pair.

Attachment of the SUMO (small ubiquitin-related modifier) to the replication factor PCNA (proliferating-cell nuclear antigen) in the budding yeast has been shown to recruit a helicase, Srs2, to active replication forks, which in turn prevents unscheduled recombination events. In the present review, I will discuss how the interaction between SUMOylated PCNA and Srs2 serves as an example for a mechanism by which SUMO modulates the properties of its targets and mediates the activation of downstream effector proteins.

Conservation of DNA damage tolerance pathways from yeast to humans.

Damage tolerance mechanisms, which allow the bypass of DNA lesions during replication, are controlled in eukaryotic cells by mono- and poly-ubiquitination of the DNA polymerase cofactor PCNA (proliferating-cell nuclear antigen). In the present review, I will summarize our current knowledge of the enzymatic machinery for ubiquitination of PCNA and the way in which the modifications affect PCNA function during replication and lesion bypass in different organisms. Using the budding yeast as a reference model, I will highlight some of the species-specific differences, but also point out the common principles that emerge from the genetic and biochemical studies of damage tolerance in a range of experimental systems.

A prokaryotic condensin/cohesin-like complex can actively compact chromosomes from a single position on the nucleoid and binds to DNA as a ring-like structure.

We show that Bacillus subtilis SMC (structural maintenance of chromosome protein) localizes to discrete foci in a cell cycle-dependent manner. Early in the cell cycle, SMC moves from the middle of the cell toward opposite cell poles in a rapid and dynamic manner and appears to interact with different regions on the chromosomes during the cell cycle. SMC colocalizes with its interacting partners, ScpA and ScpB, and the specific localization of SMC depends on both Scp proteins, showing that all three components of the SMC complex are required for proper localization. Cytological and biochemical experiments showed that dimeric ScpB stabilized the binding of ScpA to the SMC head domains. Purified SMC showed nonspecific binding to double-stranded DNA, independent of Scp proteins or ATP, and was retained on DNA after binding to closed DNA but not to linear DNA. The SMC head domains and hinge region did not show strong DNA binding activity, suggesting that the coiled-coil regions in SMC mediate an association with DNA and that SMC binds to DNA as a ring-like structure. The overproduction of SMC resulted in global chromosome compaction, while SMC was largely retained in bipolar foci, suggesting that the SMC complex forms condensation centers that actively affect global chromosome compaction from a defined position on the nucleoid.

Natural substrates of the proteasome and their recognition by the ubiquitin system.

The multitude of natural substrates of the 26S proteasome demonstrates convincingly the diversity and flexibility of the ubiquitin/proteasome system: at the same time, the number of pathways in which ubiquitin-dependent degradation is involved highlights the importance of regulated proteolysis for cellular metabolism. This review has addressed recent advances in our understanding of the principles that govern the recognition and targeting of potential substrates. While the mechanism of ubiquitin activation and conjugation is largely understood, the determination of substrate specificity by ubiquitin protein ligases remains a field of active research. Several conserved degradation signals within substrate proteins have been identified, and it is becoming increasingly clear that these serve as docking sites for specific sets of E3s, which in turn adhere to a number of well-defined strategies for the recognition of these motifs. In particular, RING finger proteins are now emerging as a new and apparently widespread class of ubiquitin ligases. The discovery of more and more E3s will undoubtedly reveal even better the common principles in architecture and mechanisms of this class of enzymes. In contrast to substrate recognition by the ubiquitin conjugation system, the way in which a ubiquitylated protein is delivered to the 26S proteasome is poorly understood. There is no doubt that multiubiquitin chains serve as the principal determinant for recognition by the proteasome, and a number of receptors and candidate targeting factors are known, some of which are associated with the proteasome itself; however, unresolved issues are the significance of the different geometries that alternatively linked multiubiquitin chains can adopt, the role of transport between subcellular compartments, as well as the participation of chaperones in the delivery step. Finally, the analysis of ubiquitin-independent, substrate-specific targeting mechanisms, such as the AZ-dependent degradation of ODC, may provide unexpected answers to questions about protein recognition by the 26S proteasome.

The srs2 suppressor of UV sensitivity acts specifically on the RAD5- and MMS2-dependent branch of the RAD6 pathway.

The SRS2 gene encodes a helicase that affects recombination, gene conversion and DNA damage repair in the yeast Saccharomyces cerevisiae. Loss-of-function mutations in srs2 suppress the extreme sensitivity towards UV radiation of rad6 and rad18 mutants, both of which are impaired in post-replication DNA repair and damage-induced mutagenesis. A sub-branch within the RAD6 pathway is mediated by RAD5, UBC13 and MMS2, and a comprehensive analysis of the srs2 effect on other known members of the RAD6 pathway reported here now demonstrates that suppression by srs2 is specific for mutants within this RAD5-dependent sub-system. Further evidence for the concerted action of RAD5 with UBC13 and MMS2 in DNA damage repair is given by examination of the effects of cell cycle stage as well as deletion of other repair systems on the activity of post-replication repair. Finally, it is shown that MMS2, like UBC13 and many other repair genes, is transcriptionally up-regulated in response to DNA damage. The data presented here support the notion that RAD5, UBC13 and MMS2 encode an ensemble of genetically and physically interacting repair factors within the RAD6 pathway that is coordinately affected by SRS2.

Activation of a membrane-bound transcription factor by regulated ubiquitin/proteasome-dependent processing.

Processing of integral membrane proteins in order to liberate active proteins is of exquisite cellular importance. Examples are the processing events that govern sterol regulation, Notch signaling, the unfolded protein response, and APP fragmentation linked to Alzheimer's disease. In these cases, the proteins are thought to be processed by regulated intramembrane proteolysis, involving site-specific, membrane-localized proteases. Here we show that two homologous yeast transcription factors SPT23 and MGA2 are made as dormant ER/nuclear membrane-localized precursors and become activated by a completely different mechanism that involves ubiquitin/proteasome-dependent processing. SPT23 and MGA2 are relatives of mammalian NF-kappaB and control unsaturated fatty acid levels. Intriguingly, proteasome-dependent processing of SPT23 is regulated by fatty acid pools, suggesting that the precursor itself or interacting partners are sensors of membrane composition or fluidity.

Two RING finger proteins mediate cooperation between ubiquitin-conjugating enzymes in DNA repair.

Two ubiquitin-conjugating enzymes, RAD6 and the heteromeric UBC13-MMS2 complex, have been implicated in post-replicative DNA damage repair in yeast. Here we provide a mechanistic basis for cooperation between the two enzymes. We show that two chromatin-associated RING finger proteins, RAD18 and RAD5, play a central role in mediating physical contacts between the members of the RAD6 pathway. RAD5 recruits the UBC13-MMS2 complex to DNA by means of its RING finger domain. Moreover, RAD5 association with RAD18 brings UBC13-MMS2 into contact with the RAD6-RAD18 complex. Interaction between the two RING finger proteins thus promotes the formation of a heteromeric complex in which the two distinct ubiquitin-conjugating activities of RAD6 and UBC13-MMS2 can be closely coordinated. Surprisingly, UBC13 and MMS2 are largely cytosolic proteins, but DNA damage triggers their redistribution to the nucleus. These findings suggest a mechanism by which the activity of this DNA repair pathway could be regulated.

A novel ubiquitination factor, E4, is involved in multiubiquitin chain assembly.

Proteins modified by multiubiquitin chains are the preferred substrates of the proteasome. Ubiquitination involves a ubiquitin-activating enzyme, E1, a ubiquitin-conjugating enzyme, E2, and often a substrate-specific ubiquitin-protein ligase, E3. Here we show that efficient multiubiquitination needed for proteasomal targeting of a model substrate requires an additional conjugation factor, named E4. This protein, previously known as UFD2 in yeast, binds to the ubiquitin moieties of preformed conjugates and catalyzes ubiquitin chain assembly in conjunction with E1, E2, and E3. Intriguingly, E4 defines a novel protein family that includes two human members and the regulatory protein NOSA from Dictyostelium required for fruiting body development. In yeast, E4 activity is linked to cell survival under stress conditions, indicating that eukaryotes utilize E4-dependent proteolysis pathways for multiple cellular functions.

Analysis of hapten binding and catalytic determinants in a family of catalytic antibodies.

We report here the cloning and kinetic analysis of a family of catalytic antibodies raised against a common transition state (TS) analog hapten, which accelerate a unimolecular oxy-Cope rearrangement. Sequence analysis revealed close homologies among the heavy chains of the catalytically active members of this set of antibodies, which derive mainly from a single germline gene, whereas the light chains can be traced back to several different, but related germline genes. The requirements for hapten binding and catalytic activity were determined by the construction of hybrid antibodies. Characterization of the latter antibodies again indicates a strong conservation of binding site structure among the catalytically active clones. The heavy chain was found to be the determining factor for catalytic efficiency, while the light chain exerted a smaller modulating effect that depended on light chain gene usage and somatic mutations. Within the heavy chain, the catalytic activity of a clone, but not hapten binding affinity, depended on the sequence of the third complementarity determining region (CDR). No correlation between high affinity for the hapten and high rate enhancement was found in the oxy-Cope system, a result that stands in contrast to the expectations from transition state theory. A mechanistic explanation for this observation is provided based on the three-dimensional crystal structure of the most active antibody, AZ-28, in complex with the hapten. This study demonstrates the utility of catalytic antibodies in examining the relationship between binding energy and catalysis in the evolution of biological catalysis, as well as expanding our understanding of the molecular basis of an immune response.

The interplay between binding energy and catalysis in the evolution of a catalytic antibody.

Antibody catalysis provides an opportunity to examine the evolution of binding energy and its relation to catalytic function in a system that has many parallels with natural enzymes. Here we report such a study involving an antibody AZ-28 that catalyses an oxy-Cope rearrangement, a pericyclic reaction that belongs to a well studied and widely used class of reactions in organic chemistry. Immunization with transition state analogue 1 results in a germline-encoded antibody that catalyses the rearrangement of hexadiene 2 to aldehyde 3 with a rate approaching that of a related pericyclic reaction catalysed by the enzyme chorismate mutase. Affinity maturation gives antibody AZ-28, which has six amino acid substitutions, one of which results in a decrease in catalytic rate. To understand the relationship between binding and catalytic rate in this system we characterized a series of active-site mutants and determined the three-dimensional crystal structure of the complex of AZ-28 with the transition state analogue. This analysis indicates that the activation energy depends on a complex balance of several stereoelectronic effects which are controlled by an extensive network of binding interactions in the active site. Thus in this instance the combinatorial diversity of the immune system provided both an efficient catalyst for a reaction where no enzyme is known, as well as an opportunity to explore the mechanisms and evolution of biological catalysis.

Antibody catalysis of pericyclic reactions.

In an effort to increase our insight into the catalysis of pericyclic reactions we have initiated a detailed study of an antibody that catalyzes an oxy-Cope rearrangement. We have determined the stereochemistry of the antibody-catalyzed reaction, and experiments are in progress to determine the conformation of the substrate bound in the antibody combining site. The genes encoding the variable regions of this antibody have been cloned and sequenced, and we have made use of a bacterial expression system to produce this antibody as a Fab fragment in recombinant form, making it amenable to genetic manipulations such as site-directed mutagenesis. The recombinant Fab fragment has been crystallized in the presence of its transition state analog, and we are now in the process of determining its active site structure.

Expression studies of catalytic antibodies.

We have examined the positive influence of human constant regions on the folding and bacterial expression of active soluble mouse immunoglobulin variable domains derived from a number of catalytic antibodies. Expression yields of eight hybridoma- and myeloma-derived chimeric Fab fragments are compared in both shake flasks and high density fermentations. In addition the usefulness of this system for the generation of in vivo expression libraries is examined by constructing and expressing combinations of heavy and light chain variable regions that were not selected as a pair during an immune response. A mutagenesis study of one of the recombinant catalytic Fab fragments reveals that single amino acid substitutions can have dramatic effects on the expression yield. This system should be generally applicable to the production of Fab fragments of catalytic and other hybridoma-derived antibodies for crystallographic and structure-function studies.

Kex2-dependent invertase secretion as a tool to study the targeting of transmembrane proteins which are involved in ER-->Golgi transport in yeast.

Mutants were isolated that are defective in the retention of a transmembrane protein in the early secretory compartments in yeast. A series of hybrid proteins was tested for their use in the selection of such mutants. Each of these hybrid proteins consisted of a type II transmembrane protein (Nin/Cout) and invertase (Suc2) as a reporter separated by a peptide linker containing a cleavage site for the Golgi protease Kex2. The integral membrane proteins which were used--Sec12p, Sec22/Sly2p or Bet1/Sly12p--are all known to be required for ER-->Golgi transport in yeast. Invertase was readily cleaved from the fusions containing Sec22/Sly2p or Bet1/Sly12p as the membrane anchoring part. In contrast, Sec12--invertase expressing transformants required mutations in either of two different genes for Kex2-dependent invertase secretion. The mutant showing the stronger retention defect (rer1) was used to clone the corresponding gene. RER1 represents the first reading frame left of the centromere of chromosome III. Cells carrying a disruption of the RER1 gene are viable and show the same mislocalizing phenotype as the original mutants. The Rer1 protein, as deduced from the nucleotide sequence, contains four transmembrane domains. It has been suggested before that Sec12p cycles between the ER and the cis-Golgi compartment. Some results obtained by using Sec12-invertase and the rer1 mutants resemble observations on the retention of Golgi-resident glycosyltransferases and viral proteins in mammalian cells. For instance, retention of Sec12-invertase is non-saturable and the membrane-spanning domain of Sec12p seems to constitute an important targeting signal.

A combinatorial approach toward DNA recognition.

A combinatorial approach has been used to identify individual RNA molecules from a large population of sequences that bind a 16-base pair homopurine-homopyrimidine DNA sequence through triple-helix formation. Fourteen of the seventeen clones selected contained stretches of pyrimidines highly homologous to the target DNA sequence (T.AT and C+.GC). In addition, these RNA molecules contained hairpin loops, interior loops, and nonstandard base triplets [C+(or C).AT, U.GC, G.GC, and A.AT] at various positions. Affinity cleavage experiments confirmed the ability of selected sequences to bind specifically to the target DNA. Systematic variation in both the target DNA sequence and buffer components should provide increased insight into the molecular interactions required for triple-helix-mediated recognition of natural DNA.