Nasmyth, O. is used by Pol to synthesize a short DNA strand, followed by elongation of the DNA strand by Pol and/or Pol?, using the short DNA fragment as a primer (43). Although the cellular counterpart of the SV40 T Betaxolol antigen has not been identified, it is believed that comparable reactions take place during chromosomal DNA replication. DNA replication in eukaryotic cells initiates from multiple replication origins that fire throughout the S phase of the cell cycle; some origins fire early, others fire late (17, 25). In and mutants (defective in cell cycle checkpoints), however, late-origin firing is not blocked by HU (40). Methyl methane sulfonate similarly inhibits late-origin firing, and this inhibition is not observed in or mutants (42). From these observations, it is proposed that this regulation of late-origin firing is usually important for the S-phase checkpoint (13, 40, 42). The gene was isolated as a multicopy suppressor of mutations in the and genes, which encode the catalytic and second-largest subunits of Pol?, respectively (5). The amino acid sequence of Dpb11 is similar to the sequence of the Cut5 (also known as Rad4) protein of (30, 37C39, 49). Both Dpb11 and Cut5 have four copies of the BRCA1 C-terminus (BRCT) domain name, Rabbit Polyclonal to C9orf89 which is thought to be an interaction domain name between proteins (7, 9, 51). In thermosensitive mutants, S-phase progression is delayed when the heat is usually shifted up, followed by cell division with unequal chromosome segregation. In the presence of HU, cells also have an elongated spindle, indicating that mitosis has started without the completion of DNA replication. Furthermore, there is a strong genetic conversation between Betaxolol Dpb11 and Pol?; high-copy suppresses the growth defects of and with one of is obtained. This suggests that Dpb11 interacts with Pol? and is required for DNA replication and the S-phase checkpoint (5). To gain a broader understanding of the function of Dpb11, we tried to identify the factors that interact with Dpb11 by isolating synthetic lethal mutations with (genes. is usually identical to the gene that encodes the third-largest subunit of Pol? (4), and is identical to (22). The gene encodes a 52-kDa protein that forms a complex with Dpb11 that is essential for DNA replication. From this analysis, we suggested that a Dpb11-Sld2 complex is required for one of the steps close to the initiation of DNA replication (27). The gene was independently isolated as the (DNA replication and checkpoint 1) gene, and the mutant was found to be defective in the S-phase checkpoint (50). To further elucidate the function of Dpb11, we analyzed the association between Dpb11, DNA polymerases, and chromatin DNA, using a chromatin immunoprecipitation (CHIP) assay (44). In the S phase of the cell cycle, Dpb11 and Pol? simultaneously associated with DNA fragments made up of an ARS, and their association was mutually dependent. We also detected a complex of Dpb11 and Pol2 that was most abundant during the S phase. Moreover, Dpb11 was required for blockage of late-origin firing by HU. Therefore, we suggest that Dpb11 is required for DNA polymerases to associate with the ARS and for blockage of late-origin firing. MATERIALS AND METHODS Plasmids. Hemagglutinin (HA) or c-epitope-tagged gene fusions were constructed by PCR amplification of the coding regions of interest and insertion of the resulting PCR products into plasmid pUC18. cassettes were inserted into the resulting plasmids, creating a C-terminal fusion with each coding sequence. An N-terminally deleted fragment of each gene fusion was subcloned into an integrating vector of the YIp family. The resulting plasmids, YIplac128-POL2-3HA/C and YIplac211-DPB11-9myc/C, were digested with ura3-1 trp1-1 leu2-3,112 his3-11,15 ade2-1 can1-100gene with a insertion mutant allele and subsequently popping out the gene. W303-1Ab (were constructed Betaxolol by a homologous recombination that disrupted the wild-type allele and simultaneously created a C-terminal epitope-tagged version of the.