No abstract

Proliferating cell nuclear antigen (PCNA) is essential for eukaryotic DNA replication and functions as a processivity factor of DNA polymerase ␦ (pol ␦). Due to the functional and structural similarity with the ␤-subunit of Escherichia coli DNA polymerase III, it has been proposed that PCNA would act as a molecular clamp during DNA synthesis. By site-directed mutagenesis and biochemical analyses, we have studied the functional domains of human PCNA required for stimulation of replication factor C (RF-C) ATPase and DNA synthesis by pol ␦. Short deletions from either the N or C termini caused drastic changes in extraction and chromatographic behaviors, suggesting that both of these terminal regions are crucial to fold the tertiary structure of PCNA. The short C-terminal stretch from Lys 254 to Glu 256 is necessary for stimulation of RF-C ATPase activity, but not for stimulation of DNA synthesis by pol ␦. Nine basic amino acids that are essential for activating DNA synthesis by pol ␦ are positioned at the internal ␣-helices of PCNA. This result is in good agreement with the observation that PCNA has a ring structure similar to the ␤-subunit and clamps a template DNA through this positively charged internal surface. Several other charged amino acids are also required to stimulate either RF-C ATPase or pol ␦ DNA synthesis. Some of them are positioned at loops which are exposed on one of the side surface of PCNA adjacent to the C-terminal loop. In addition, the ␤-sheets composing the intermolecular interface of the trimeric PCNA are important for interaction with pol ␦. Therefore, the outer surface of PCNA has multiple functional surfaces which are responsible for the interaction with multiple factors. Furthermore, the two side surfaces seem to be functionally distinguishable, and this may determine the orientation of tracking PCNA along the DNA.The proliferating cell nuclear antigen (PCNA) 1 is an essential replication factor for simian virus 40 (SV40) DNA replication in vitro and is involved in the elongation stages of DNA replication (1-4). Recent studies have further demonstrated the involvement of PCNA in cellular chromosomal DNA replication in vivo (5-7). In addition to its essential role in DNA replication, PCNA is required for nucleotide excision repair of DNA (8 -10) and also may participate in the cell cycle control as demonstrated by an interaction with a cyclin dependent kinase complex (11). Therefore, PCNA is multifunctional through the interaction with several specific partners, and all of the functions are crucial for cell proliferation.Genes encoding PCNA have been isolated from various eukaryotes and are composed of highly conserved amino acid sequences of around 260 residues. The functions of PCNA during DNA replication have been elucidated by studies of SV40 DNA replication in vitro. In this reaction, three protein components, PCNA, DNA polymerase (pol) ␦, and replication factor C (RF-C) are required for leading strand DNA synthesis following the initiation of DNA synthesis by DNA pol ␣ at the SV4...

Background: PCNA, an eukaryotic DNA sliding clamp interacts with replication factors and the cell cycle protein, p21(Cip1/Waf1) and functions as a molecular switch for DNA elongation. To understand how DNA replication is regulated through PCNA, elucidation of the precise mechanisms of these protein interactions is necessary.

Background: Ischemic preconditioning (IPC) is an effective procedure to protect against ischemia/reperfusion (I/R) injury. Hypoxiainducible factor-1α (Hif-1α) is a key molecule in IPC, and roxadustat (RXD), a first-in-class prolyl hydroxylase domain-containing protein inhibitor, has been recently developed to treat anemia in patients with chronic kidney disease. Thus, we investigated whether RXD pretreatment protects against I/R injury. Methods and Results: RXD pretreatment markedly reduced the infarct size and suppressed plasma creatinine kinase activity in a murine I/R model. Analysis of oxygen metabolism showed that RXD could produce ischemic tolerance by shifting metabolism from aerobic to anaerobic respiration.Conclusions: RXD pretreatment may be a novel strategy against I/R injury.

No abstract