The inner side of yeast PCNA contributes to genome stability by mediating interactions with Rad18 and the replicative DNA polymerase δ.

PCNA is a central orchestrator of cellular processes linked to DNA metabolism. It is a binding platform for a plethora of proteins and coordinates and regulates the activity of several pathways. The outer side of PCNA comprises most of the known interacting and regulatory surfaces, whereas the residues at the inner side constitute the sliding surface facing the DNA double helix. Here, by investigating the L154A mutation found at the inner side, we show that the inner surface mediates protein interactions essential for genome stability. It forms part of the binding site of Rad18, a key regulator of DNA damage tolerance, and is required for PCNA sumoylation which prevents unscheduled recombination during replication. In addition, the L154 residue is necessary for stable complex formation between PCNA and the replicative DNA polymerase δ. Hence, its absence increases the mutation burden of yeast cells due to faulty replication. In summary, the essential role of the L154 of PCNA in guarding and maintaining stable replication and promoting DNA damage tolerance reveals a new connection between these processes and assigns a new coordinating function to the central channel of PCNA.

The Zn-finger of Saccharomyces cerevisiae Rad18 and its adjacent region mediate interaction with Rad5.

DNA damages that hinder the movement of the replication complex can ultimately lead to cell death. To avoid that, cells possess several DNA damage bypass mechanisms. The Rad18 ubiquitin ligase controls error-free and mutagenic pathways that help the replication complex to bypass DNA lesions by monoubiquitylating PCNA at stalled replication forks. In Saccharomyces cerevisiae, two of the Rad18 governed pathways are activated by monoubiquitylated PCNA and they involve translesion synthesis polymerases, whereas a third pathway needs subsequent polyubiquitylation of the same PCNA residue by another ubiquitin ligase the Rad5 protein, and it employs template switching. The goal of this study was to dissect the regulatory role of the multidomain Rad18 in DNA damage bypass using a structure-function based approach. Investigating deletion and point mutant RAD18 variants in yeast genetic and yeast two-hybrid assays we show that the Zn-finger of Rad18 mediates its interaction with Rad5, and the N-terminal adjacent region is also necessary for Rad5 binding. Moreover, results of the yeast two-hybrid and in vivo ubiquitylation experiments raise the possibility that direct interaction between Rad18 and Rad5 might not be necessary for the function of the Rad5 dependent pathway. The presented data also reveal that yeast Rad18 uses different domains to mediate its association with itself and with Rad5. Our results contribute to better understanding of the complex machinery of DNA damage bypass pathways.

Single Cell Mass Cytometry of Non-Small Cell Lung Cancer Cells Reveals Complexity of In vivo And Three-Dimensional Models over the Petri-dish.

Single cell genomics and proteomics with the combination of innovative three-dimensional (3D) cell culture techniques can open new avenues toward the understanding of intra-tumor heterogeneity. Here, we characterize lung cancer markers using single cell mass cytometry to compare different in vitro cell culturing methods: two-dimensional (2D), carrier-free, or bead-based 3D culturing with in vivo xenografts. Proliferation, viability, and cell cycle phase distribution has been investigated. Gene expression analysis enabled the selection of markers that were overexpressed: TMEM45A, SLC16A3, CD66, SLC2A1, CA9, CD24, or repressed: EGFR either in vivo or in long-term 3D cultures. Additionally, TRA-1-60, pan-keratins, CD326, Galectin-3, and CD274, markers with known clinical significance have been investigated at single cell resolution. The described twelve markers convincingly highlighted a unique pattern reflecting intra-tumor heterogeneity of 3D samples and in vivo A549 lung cancer cells. In 3D systems CA9, CD24, and EGFR showed higher expression than in vivo. Multidimensional single cell proteome profiling revealed that 3D cultures represent a transition from 2D to in vivo conditions by intermediate marker expression of TRA-1-60, TMEM45A, pan-keratin, CD326, MCT4, Gal-3, CD66, GLUT1, and CD274. Therefore, 3D cultures of NSCLC cells bearing more putative cancer targets should be used in drug screening as the preferred technique rather than the Petri-dish.

Mutations at the Subunit Interface of Yeast Proliferating Cell Nuclear Antigen Reveal a Versatile Regulatory Domain.

Proliferating cell nuclear antigen (PCNA) plays a key role in many cellular processes and due to that it interacts with a plethora of proteins. The main interacting surfaces of Saccharomyces cerevisiae PCNA have been mapped to the interdomain connecting loop and to the carboxy-terminal domain. Here we report that the subunit interface of yeast PCNA also has regulatory roles in the function of several DNA damage response pathways. Using site-directed mutagenesis we engineered mutations at both sides of the interface and investigated the effect of these alleles on DNA damage response. Genetic experiments with strains bearing the mutant alleles revealed that mutagenic translesion synthesis, nucleotide excision repair, and homologous recombination are all regulated through residues at the subunit interface. Moreover, genetic characterization of one of our mutants identifies a new sub-branch of nucleotide excision repair. Based on these results we conclude that residues at the subunit boundary of PCNA are not only important for the formation of the trimer structure of PCNA, but they constitute a regulatory protein domain that mediates different DNA damage response pathways, as well.

Def1 promotes the degradation of Pol3 for polymerase exchange to occur during DNA-damage--induced mutagenesis in Saccharomyces cerevisiae.

DNA damages hinder the advance of replication forks because of the inability of the replicative polymerases to synthesize across most DNA lesions. Because stalled replication forks are prone to undergo DNA breakage and recombination that can lead to chromosomal rearrangements and cell death, cells possess different mechanisms to ensure the continuity of replication on damaged templates. Specialized, translesion synthesis (TLS) polymerases can take over synthesis at DNA damage sites. TLS polymerases synthesize DNA with a high error rate and are responsible for damage-induced mutagenesis, so their activity must be strictly regulated. However, the mechanism that allows their replacement of the replicative polymerase is unknown. Here, using protein complex purification and yeast genetic tools, we identify Def1 as a key factor for damage-induced mutagenesis in yeast. In in vivo experiments we demonstrate that upon DNA damage, Def1 promotes the ubiquitylation and subsequent proteasomal degradation of Pol3, the catalytic subunit of the replicative polymerase δ, whereas Pol31 and Pol32, the other two subunits of polymerase δ, are not affected. We also show that purified Pol31 and Pol32 can form a complex with the TLS polymerase Rev1. Our results imply that TLS polymerases carry out DNA lesion bypass only after the Def1-assisted removal of Pol3 from the stalled replication fork.