Ubiquitinated proliferating cell nuclear antigen activates translesion DNA polymerases eta and REV1

In response to DNA damage, the Rad6/Rad18 ubiquitin-conjugating complex monoubiquitinates the replication clamp proliferating cell nuclear antigen (PCNA) at Lys-164. Although ubiquitination of PCNA is recognized as an essential step in initiating postreplication repair, the mechanistic relevance of this modification has remained elusive. Here, we describe a robust in vitro system that ubiquitinates yeast PCNA specifically on Lys-164. Significantly, only those PCNA clamps that are appropriately loaded around effector DNA by its loader, replication factor C, are ubiquitinated. This observation suggests that, in vitro, only PCNA present at stalled replication forks is ubiquitinated. Ubiquitinated PCNA displays the same replicative functions as unmodified PCNA. These functions include loading onto DNA by replication factor C, as well as Okazaki fragment synthesis and maturation by the PCNA-coordinated actions of DNA polymerase delta, the flap endonuclease FEN1, and DNA ligase I. However, whereas the activity of DNA polymerase zeta remains unaffected by ubiquitination of PCNA, ubiquitinated PCNA specifically activates two key enzymes in translesion synthesis: DNA polymerase eta, the yeast Xeroderma pigmentosum ortholog, and Rev1, a deoxycytidyl transferase that functions in organizing the mutagenic DNA replication machinery. We propose that ubiquitination of PCNA increases its functionality as a sliding clamp to promote mutagenic DNA replication.     

The effects of recombinant-DNA-derived interferons on the growth of myeloid progenitor cells

Interferons (IFNs) have been shown to have significant effects on hematopoietic cell growth. Previous studies defining these effects have utilized mouse and human alpha-, beta-, and gamma-IFN isolated from supernatants of stimulated cells. Despite purification, the possible presence of other lymphokines and soluble factors remains a concern. In this study, the effects of gene-cloned alpha- and gamma-IFN on colony- forming units of granulocyte/macrophage (CFU-GM) progenitors cultured from the peripheral blood of normal volunteers were examined. In addition, blast cell colonies from one patient with acute myelogenous leukemia (AML) were studied. The growth of normal CFU-GM and AML blast cell colonies was inhibited in a dose-dependent manner by gamma- and alpha-IFN. gamma-IFN was ten to 100 times more potent than alpha-IFN in that this species of IFN reduced colony formation by greater than 50% at concentrations of less than 15 antiviral U/mL. The effects of gamma- IFN were neutralized by a monoclonal antibody specific for gamma-IFN. These in vitro studies indicate that human gamma-IFN may be an important modulator of myelopoiesis. Although these data indicate a possible efficacy of gamma-IFN in the treatment of AML, the in vitro results should be considered for their in vivo significance.     

Requirement of proliferating cell nuclear antigen in RAD6-dependent postreplicational DNA repair.

The proliferating cell nuclear antigen (PCNA) acts as a processivity factor for replicative DNA polymerases and is essential for DNA replication. In vitro studies have suggested a role for PCNA-in the repair synthesis step of nucleotide excision repair, and PCNA interacts with the cyclin-dependent kinase inhibitor p21. However, because of the lack of genetic evidence, it is not clear which of the DNA repair processes are in fact affected by PCNA in vivo. Here, we describe a PCNA mutation, pol30-46, that confers ultraviolet (UV) sensitivity but has no effect on growth or cell cycle progression, and the mutant pcna interacts normally with DNA polymerase delta and epsilon. Genetic studies indicate that the pol30-46 mutation is specifically defective in RAD6-dependent postreplicational repair of UV damaged DNA, and this mutation impairs the error-free mode of bypass repair. These results implicate a role for PCNA as an intermediary between DNA replication and postreplicational DNA repair.     

Sequential switching of binding partners on PCNA during in vitro Okazaki fragment maturation

The homotrimeric sliding clamp proliferating cell nuclear antigen (PCNA) mediates Okazaki fragment maturation through tight coordination of the activities of DNA polymerase δ (Pol δ), flap endonuclease 1 (FEN1) and DNA ligase I (Lig1). Little is known regarding the mechanism of partner switching on PCNA and the involvement of PCNA''s three binding sites in coordinating such processes. To shed new light on PCNA-mediated Okazaki fragment maturation, we developed a novel approach for the generation of PCNA heterotrimers containing one or two mutant monomers that are unable to bind and stimulate partners. These heterotrimers maintain the native oligomeric structure of PCNA and exhibit high stability under various conditions. Unexpectedly, we found that PCNA heterotrimers containing only one functional binding site enable Okazaki fragment maturation by efficiently coordinating the activities of Pol δ, FEN1, and Lig1. The efficiency of switching between partners on PCNA was not significantly impaired by limiting the number of available binding sites on the PCNA ring. Our results provide the first direct evidence, to our knowledge, that simultaneous binding of multiple partners to PCNA is unnecessary, and if it occurs, does not provide significant functional advantages for PCNA-mediated Okazaki fragment maturation in vitro. In contrast to the “toolbelt” model, which was demonstrated for bacterial and archaeal sliding clamps, our results suggest a mechanism of sequential switching of partners on the eukaryotic PCNA trimer during DNA replication and repair.Proliferating cell nuclear antigen (PCNA) is a central coordinator of genome duplication and maintenance pathways in eukaryotes (1, 2). A member of the conserved sliding clamp family, PCNA is a homotrimeric ring-shaped protein that encircles DNA and serves as a processivity factor for DNA polymerases and a binding platform for many DNA modifying enzymes. PCNA interacts with partners involved in numerous processes, including DNA replication, recombination and repair, chromatin remodeling, and cell-cycle regulation. PCNA recruits these partners to replication forks or other chromosomal locations, enhances their catalytic activities, and orchestrates their cooperation in multistep enzymatic processes. Because most partners interact with the same binding site on PCNA, competition for binding must be tightly regulated during complex PCNA-mediated processes. The switching of partners on the PCNA platform has been shown to be crucial for the proper progression of multiple DNA replication and repair pathways, such as lagging strand replication, translesion synthesis, and mismatch repair (1). In recent years, several regulatory mechanisms, mostly involving posttranslational modifications of PCNA by ubiquitin or small ubiquitin-like modifier, have been shown to affect partner switching on PCNA by favoring the recruitment of specific partners (3–5).Despite extensive research into the regulation of PCNA-mediated processes, very little is known regarding how PCNA coordinates the activity of several enzymes during sequential processes. Two simple models have been proposed to explain this coordination (1, 2, 6, 7). The first model assumes highly dynamic partner switching on PCNA due to sequential binding and release events on the same or different PCNA monomers (Fig. 1, Upper). This model predicts that a single functional binding site on the PCNA trimer should be sufficient for the coordination of the entire process. In contrast, the second model assumes simultaneous binding of two or three partners to different monomers on the PCNA trimer (Fig. 1, Lower). In this case, the partners are stably associated with PCNA, which acts as a “toolbelt” throughout the process. According to this model, only PCNA trimers with two or three functional binding sites would be able to coordinate the process.Open in a separate windowFig. 1.Two possible models describing PCNA-mediated Okazaki fragment maturation. (Upper) A dynamic model in which Pol δ, FEN1, and Lig1 are bound and released from PCNA in a sequential manner. (Lower) The toolbelt model in which the three enzymes are simultaneously bound to PCNA using all available PCNA binding sites. The red segments represent the RNA primers; glowing circles represent enzymes currently active on the substrate.One of the best studied examples of such a multipartner PCNA-mediated process is the synthesis and maturation of Okazaki fragments during lagging strand DNA replication. This process involves the sequential activity of three PCNA binding partners—DNA polymerase δ (Pol δ), flap endonuclease 1 (FEN1), and DNA ligase I (Lig1), which mediate DNA synthesis, flap cleavage, and ligation, respectively (7–9). This is a fast and efficient process that is estimated to take place ∼100,000 times during each yeast cell division with a low tolerance for errors (8). The enzymes involved must cooperate through PCNA in a tightly regulated manner, acting sequentially on the same substrate while repeatedly exchanging access to it (Fig. 1). In particular, removal of the initiator RNA requires several rapid iterative switches between Pol δ and FEN1 (7). This PCNA-dependent cooperation is particularly important to ensure that flaps will not become too long for processing by this short-flap pathway (7, 10, 11).To directly examine the mechanism of partner switching on PCNA and the functional significance of its homotrimeric structure, we developed a novel approach for the generation of PCNA heterotrimers that contain both wild-type (WT) and mutant monomers that are unable to bind different partners. We used these heterotrimers to determine whether simultaneous binding of more than one partner to a PCNA trimer is necessary to coordinate PCNA-mediated nick translation and Okazaki fragment maturation. Contrary to the toolbelt model, our findings indicate that simultaneous binding is not required, and sequential switching of partners on a single monomer of PCNA is sufficient to coordinate Okazaki fragment maturation. Our findings suggest that PCNA can efficiently orchestrate complex processes by regulating sequential binding and release events of several partners without binding them simultaneously.     

Late radiation injuries of the gastrointestinal tract in the H2 and H5 EORTC Hodgkin's disease trials: emphasis on the role of exploratory laparotomy and fractionation

Out of 516 patients who entered in the two successive EORTC trials H2 and H5 for supra-diaphragmatic stages I and II Hodgkin's disease (HD), and who received an infra-diaphragmatic irradiation, 36 (7%) developed late radiation injuries of the gastrointestinal tract (GIT). Twenty-five patients presented with ulcers (stomach or duodenum), 2 with severe gastritis, 6 with small bowel obstruction or perforation and 3 patients had both an ulcer and bowel obstruction. A previous laparotomy played an important role. While the complication rate was 2.7% without any previous abdominal surgery, it was 11.5% after laparotomy (p less than 0.001). Fractionation was also found to be of importance in the occurrence of complications: three different weekly schedules were used -5 x 2 Gy, 4 x 2.5 Gy and 3 x 3.3 Gy; the GIT complication rates were 4, 9 and 22%, respectively (p less than 0.001). When combining laparotomy and fractionation, we found that the patients who were treated using 5 weekly fractions of 2 Gy without any prior laparotomy had a very low rate of late digestive complications (1%), whereas the patients who received 3 weekly fractions of 3.3 Gy after laparotomy presented a 39% complication rate. The other subgroups of patients were at an intermediate risk (from 5 to 13%) of late digestive injuries. Since most patients received 40 Gy with only very small variations, the influence of the radiation dose could not be investigated.(ABSTRACT TRUNCATED AT 250 WORDS)