Linkage reprogramming by tailor-made E3s reveals polyubiquitin chain requirements in DNA-damage bypass.

A polyubiquitin chain can adopt a variety of shapes, depending on how the ubiquitin monomers are joined. However, the relevance of linkage for the signaling functions of polyubiquitin chains is often poorly understood because of our inability to control or manipulate this parameter in vivo. Here, we present a strategy for reprogramming polyubiquitin chain linkage by means of tailor-made, linkage- and substrate-selective ubiquitin ligases. Using the polyubiquitylation of the budding yeast replication factor PCNA in response to DNA damage as a model case, we show that altering the features of a polyubiquitin chain in vivo can change the fate of the modified substrate. We also provide evidence for redundancy between distinct but structurally similar linkages, and we demonstrate by proof-of-principle experiments that the method can be generalized to targets beyond PCNA. Our study illustrates a promising approach toward the in vivo analysis of polyubiquitin signaling.

Topoisomerase I (TOP1) dynamics: conformational transition from open to closed states.

Eukaryotic topoisomerases I (TOP1) are ubiquitous enzymes removing DNA torsional stress. However, there is little data concerning the three-dimensional structure of TOP1 in the absence of DNA, nor how the DNA molecule can enter/exit its closed conformation. Here, we solved the structure of thermostable archaeal Caldiarchaeum subterraneum CsTOP1 in an apo-form. The enzyme displays an open conformation resulting from one substantial rotation between the capping (CAP) and the catalytic (CAT) modules. The junction between these two modules is a five-residue loop, the hinge, whose flexibility permits the opening/closing of the enzyme and the entry of DNA. We identified a highly conserved tyrosine near the hinge as mediating the transition from the open to closed conformation upon DNA binding. Directed mutagenesis confirmed the importance of the hinge flexibility, and linked the enzyme dynamics with sensitivity to camptothecin, a TOP1 inhibitor targeting the TOP1 enzyme catalytic site in the closed conformation.

Readers of PCNA modifications.

The eukaryotic sliding clamp, proliferating cell nuclear antigen (PCNA), acts as a central coordinator of DNA transactions by providing a multivalent interaction surface for factors involved in DNA replication, repair, chromatin dynamics and cell cycle regulation. Posttranslational modifications (PTMs), such as mono- and polyubiquitylation, sumoylation, phosphorylation and acetylation, further expand the repertoire of PCNA's binding partners. These modifications affect PCNA's activity in the bypass of lesions during DNA replication, the regulation of alternative damage processing pathways such as homologous recombination and DNA interstrand cross-link repair, or impact on the stability of PCNA itself. In this review, we summarise our current knowledge about how the PTMs are "read" by downstream effector proteins that mediate the appropriate action. Given the variety of interaction partners responding to PCNA's modified forms, the ensemble of PCNA modifications serves as an instructive model for the study of biological signalling through PTMs in general.

Topoisomerase 1 provokes the formation of short deletions in repeated sequences upon high transcription in Saccharomyces cerevisiae.

High transcription is associated with genetic instability, notably increased spontaneous mutation rates, which is a phenomenon termed Transcription-Associated-Mutagenesis (TAM). In this study, we investigated TAM using the chromosomal CAN1 gene under the transcriptional control of two strong and inducible promoters (pGAL1 and pTET) in Saccharomyces cerevisiae. Both pTET- and pGAL1-driven high transcription at the CAN1 gene result in enhanced spontaneous mutation rates. Comparison of both promoters reveals differences in the type of mutagenesis, except for short (-2 and -3 nt) deletions, which depend only on the level of transcription. This mutation type, characteristic of TAM, is sequence dependent, occurring prefentially at di- and trinucleotides repeats, notably at two mutational hotspots encompassing the same 5'-ACATAT-3' sequence. To explore the mechanisms underlying the formation of short deletions in the course of TAM, we have determined Can(R) mutation spectra in yeast mutants affected in DNA metabolism. We identified topoisomerase 1-deficient strains (top1Δ) that specifically abolish the formation of short deletions under high transcription. The rate of the formation of (-2/-3nt) deletions is also reduced in the absence of RAD1 and MUS81 genes, involved in the repair of Top1p-DNA covalent complex. Furthermore ChIP analysis reveals an enrichment of trapped Top1p in the CAN1 ORF under high transcription. We propose a model, in which the repair of trapped Top1p-DNA complexes provokes the formation of short deletion in S. cerevisiae. This study reveals unavoidable conflicts between Top1p and the transcriptional machinery and their potential impact on genome stability.