The immature plasmacytoid dendritic cell (PDC) is identical with the principal type I IFN‐producing cell upon viral infection. Oligodeoxynucleotides which contain unmethylated CpG motifs (CpG ODN) are recognized by the vertebrate immune system. Previously, we described CpG ODN that strongly activate human B cells and human blood dendritic cells. Here we describe distinct CpG‐containing oligonucleotide sequences which, in contrast to previously described CpG ODN, induced high amounts of IFN‐α and IFN‐β in peripheral blood mononuclear cells (PBMC). Intracellular staining for IFN‐α revealed that within PBMC CpG ODN‐induced IFN‐α is produced exclusively by PDC. Unlike IFN‐α, TNF‐α is up‐regulated in PDC by all CpG ODN tested. Purified PDC responded to CpG ODN, demonstrating direct activation of PDC by CpG ODN. The most active sequence induced the production of up to 5 pg IFN‐α per single PDC, resulting in more than 400 ng/ml IFN‐α in the supernatant of PBMC enriched for PDC. The potency of CpG ODN to stimulate IFN‐α correlated with their ability to stimulate NK cell lytic activity, while purified NK cells did not respond to CpG ODN. IFNγ production in PBMC was dependent on CpG ODN‐induced IFN‐α/β as demonstrated by IFN‐α/β blocking antibodies. IFN‐α‐inducing CpG ODN strongly supported IFN‐γ production of TCR‐triggered CD4 T cells but were less active than other CpG ODN in stimulating B cells. In conclusion our results demonstrate that particular CpG ODN sequences exist which, due to high IFN‐α/β induction in PDC, induce a set of immune responses typical for viral infection.

DNA damage tolerance (DDT) is responsible for genomic stability and cell viability by bypassing the replication block. In Saccharomyces cerevisiae DDT employs two parallel branch pathways to bypass the DNA lesion, namely translesion DNA synthesis (TLS) and error-free lesion bypass, which are mediated by sequential modifications of PCNA. Rad5 has been placed in the error-free branch of DDT because it contains an E3 ligase domain required for PCNA polyubiquitination. Rad5 is a multi-functional protein and may also play a role in TLS, since it interacts with the TLS polymerase Rev1. In this study we mapped the Rev1-interaction domain in Rad5 to the amino acid resolution and demonstrated that Rad5 is indeed involved in TLS possibly through recruitment of Rev1. Genetic analyses show that the dual functions of Rad5 can be separated and reconstituted. Crystal structure analysis of the Rad5–Rev1 interaction reveals a consensus RFF motif in the Rad5 N-terminus that binds to a hydrophobic pocket within the C-terminal domain of Rev1 that is highly conserved in eukaryotes. This study indicates that Rad5 plays a critical role in pathway choice between TLS and error-free DDT.

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Spontaneous mutations occur in the DNA as a result of endogenous cellular processes. The antimutagenic processes within a cell consist primarily of mechanisms of DNA repair, which are critical for maintenance of genomic stability, while mutagenic processes include mistakes by the replicative machinery and spontaneous alterations in the base chemistry of DNA. In Saccharomyces cerevisiae spontaneous mutagenesis assays are typically employed when studying the DNA damage repair pathways, since loss of one of these mechanisms results in a detectable increase in the spontaneous mutation rate, which is determined by first growing cells to log phase, then subculturing them to a very low concentration and incubating for several days. This allows for many cell divisions and thus many opportunities for mutations to occur in the genome. The selection of mutants is typically based on a specific genetic marker such as an auxotrophic marker, and the total number is compared to the total number of viable cells in order to determine the mutation rate for an exponentially growing culture.