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ABSIRATCA target protein for nalidixic and oxolinic acids in Escherichia coli, the nalA gene product (Pnal), was purified to homogeneity as judged by gel electrophoresis, using an in vitro complementation assay. It is a dimer of identical 110,000-dalton subunits. A (2), and ColEl DNA (8) were prepared as described. Relaxed kX174 RF and ColEl DNA were prepared either by nicking with pancreatic DNase followed by sealing with T4 DNA ligase (9) or by relaxation with E. coli w protein (6).Enzyme Assays. The Pnal assay, which will be detailed elsewhere, was essentially that devised by C. Sumida-Yasumoto using a nalAr strain constructed by R. Sternglanz. It is based on the dominant conferral of drug sensitivity by addition of wild-type protein to a 4X174 RFI replication system (2) directed by H560-1 extracts. The reaction mixtures (0.05 ml) contained 1 nmol of OXX174 RFI, 0.1 mg of H560-1 receptor (NH4)2SO4 fraction, 10 jig of Fraction II prepared from H560 infected with 4X174 am3 Pnal, and 30 ,ug/ml of Oxo or 100 ,gg/ml of Nal. One unit of Pnal catalyzes the sensitization of 1 nmol of dTMP incorporation to Nal or Oxo in 30 min at 300.The DNA gyrase assay measures the conversion of relaxed ColEl DNA to the supercoiled form as monitored by agarose gel electrophoresis (3). The reaction mixture (35 ,l) contained 35 mM Tris-HCl at pH 7.6, 6 mM MgCl2, 18 mM potassium phosphate, 5 mM spermidine-HCl, 1.4 mM ATP, yeast tRNA at 90 sg/ml, 5 mM dithiothreitol, bovine serum albumin at 50 Mg/1ml, 0.2 Mg of relaxed DNA, and enzyme. After 60 min at 300, 10 Al of a 25% (vol/vol) glycerol solution containing bromophenol blue at 0.25 mg/ml and either 5% sodium dodecyl sulfate (NaDodSO4), 2.5% Sarkosyl, or 45 mM EDTA was added. The mixture was applied to a 13 X 15 X 0.4 cm slab gel of 1.0% agarose and then subjected to electrophoresis at 40 V for [14][15][16] hr at 230 (3). The gels were stained, destained, and photographed using shortwave ultraviolet light (3). Negatives were traced with a Joyce-Loebl microdensitometer. One unit of DNA gyrase converts 0.1 Mug of relaxed DNA to the superAbbreviations: Nal, nalidixic acid; Oxo, oxolinic acid; Pnal, Nal target protein; N-C, nicking-closing; RF, replicative form; NaDodSO4, sodium dodecyl sulfate. An allele conferring resistance to a drug is indicated by r.

Eukaryotic chromosomal DNA replication requires a two-step assembly of replication proteins on origins; formation of the prereplicative complex (pre-RC) in late M and G1 phases of the cell cycle, and assembly of other replication proteins in S phase to load DNA polymerases to initiate DNA synthesis. In budding yeast, assembly of Dpb11 and the Sld3-Cdc45 complex on the pre-RC at origins is required for loading DNA polymerases. Here we describe a novel replication complex, GINS (Go, Ichi, Nii, and San; five, one, two, and three in Japanese), in budding yeast, consisting of Sld5, Psf1 (partner of Sld five 1), Psf2, and Psf3 proteins, all of which are highly conserved in eukaryotic cells. Since the conditional mutations of Sld5 and Psf1 confer defect of DNA replication under nonpermissive conditions, GINS is suggested to function for chromosomal DNA replication. Consistently, in S phase, GINS associates first with replication origins and then with neighboring sequences. Without GINS, neither Dpb11 nor Cdc45 associates properly with chromatin DNA. Conversely, without Dpb11 or Sld3, GINS does not associate with origins. Moreover, genetic and two-hybrid interactions suggest that GINS interacts with Sld3 and Dpb11. Therefore, Dpb11, Sld3, Cdc45, and GINS assemble in a mutually dependent manner on replication origins to initiate DNA synthesis.[Keywords: DNA replication; replication protein; Dpb11; Sld3; Sld5] Supplemental material is available at http://www.genesdev.org.

No abstract

Mutation of predicted 3′‐‐>5′ exonuclease active site residues of Saccharomyces cerevisiae POL3 DNA polymerase (delta) or deletion of the PMS1 mismatch repair gene lead to relative (to wild type) spontaneous mutation rates of approximately 130 and 41, respectively, measured at a URA3 reporter gene inserted near to a defined replication origin. The POL3 exonuclease‐deficient mutant pol3‐01 generated most classes of single base mutation in URA3, indicating a broad specificity that generally corresponds to that of the PMS1 system. pol3‐01 pms1 haploid cells ceased growth after a few divisions with no unique terminal cell morphology. A pol3‐01/pol3‐01 pms1/pms1 diploid was viable and displayed an estimated URA3 relative mutation rate of 2 × 10(4), which we calculate to be catastrophically high in a haploid. The relationship between the relative mutation rates of pol3‐01 and pms1 was multiplicative, indicating action in series. The PMS1 transcript showed the same cell cycle periodicity as those of a set of DNA replication genes that includes POL3, suggesting PMS1 is co‐regulated with these genes. We propose that the POL3 3′‐‐>5′ exonuclease and the PMS1 mismatch repair system act on a common pathway analogous to the dnaQ‐‐>mutHLS pathway of DNA replication error correction in Escherichia coli.