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Mol Cell Biol 15 3813 1995 37 R Rothstein Methods Enzymol 194 281 1991 38 N Valtz and M Peter ibid 283 350 1997 39 E Harlow and D Lane Antibodies A Laboratory Manual Cold Spring Harbor Laboratory Press Cold Spring Harbor NY 1988 40 The plasmid ACB435 expressing full length Far1p from the GAL promoter was constructed as follows plasmid pTP62 22 was digested with Bam HI the 1910 bp fragment was puri ed and ligated together with the 582 bp Bam HI Xho I fragment isolated from plasmid ACB414 20 into pRS315 G R S Sikorski and P Hieter Genetics 122 19 1989 linearized with Bam HI and Xho I Expression of the Far1 protein was controlled by immunoblotting with Far1p antibodies A single copy of the HA epitope was fused to the NH2 terminus of CDC24 using PCR and the fragment was cloned into the vector p425 ADH D Mumberg R Mu ller M Funk Gene 156 119 1995 which allows expression of Cdc24p Localization of Bacterial DNA Polymerase Evidence for a Factory Model of Replication Katherine P Lemon and Alan D Grossman Two general models have been proposed for DNA replication In one model DNA polymerase moves along the DNA like a train on a track in the other model the polymerase is stationary like a factory and DNA is pulled through To distinguish between these models we visualized DNA polymerase of the bacterium Bacillus subtilis in living cells by the creation of a fusion protein containing the catalytic subunit PolC and green uorescent protein GFP PolC GFP was localized at discrete intracellular positions predominantly at or near midcell rather than being distributed randomly These results suggest that the polymerase is anchored in place and thus support the model in which the DNA template moves through the polymerase For all organisms the production of viable progeny depends on the faithful replication of DNA by DNA polymerase A conceptual question about in vivo DNA replication remains unsettled During replication does the DNA polymerase move along the DNA template or is the DNA polymerase in a fixed position with the DNA template moving through the replication machinery Studies with eukaryotic cells have indicated that DNA replication proteins and newly replicated DNA are present at numerous discrete foci so called replication factories 1 resulting in the hypothesis that DNA replication occurs at fixed locations However analysis of eukaryotic DNA polymerase is complicated because replication starts from many different origins and because it is difficult to orient the foci within the eukaryotic nucleus Department of Biology Building 68 530 Massachusetts Institute of Technology Cambridge MA 02139 USA To whom correspondence should be addressed Email adg mit edu 1516 Like many bacteria Bacillus subtilis has a single circular chromosome 4200 kilobase pairs 2 and DNA replication initiates from a single origin oriC and proceeds bidirectionally 3 Most of the proteins present at the replication fork are conserved in prokaryotes and eukaryotes 4 We attempted to determine whether in a population of cells at different stages of the replication cycle the replicative DNA polymerase of B subtilis functions at fixed intra from the constitutive ADH promoter The plasmid fully restores viability of a temperature sensitive cdc24 5 strain at 37 C 41 A C Butty and M Peter data not shown 42 We thank members of each laboratory for helpful discussions C Boone R A Arkowitz J Chant D Lew M Funk E Elion E O Shea H O Park E Bi and E Leberer for plasmids and strains B Catarin for polyclonal antibodies to Far1p and E Leberer for antibodies to Bem1p We also thank N Valtz S Henchoz and K Peter for help during early aspects of this work and J Philips V Simanis and R Iggo for critical reading of the manuscript L S H was supported by an NIH postdoctoral fellowship Work in the I H laboratory was supported by an NIH research grant GM48052 P M P is supported by grants from the Worcester Foundation the Millipore Foundation and NIH GM57769 M P is supported by the Swiss National Science Foundation the Swiss Cancer League and a Helmut Horten Incentive award I H dedicates this paper to J Stahl 9 June 1998 accepted 16 October 1998 cellular positions or if it is randomly distributed along the nucleoid We visualized DNA polymerase in living cells using a fusion protein consisting of the catalytic subunit PolC attached in frame to green fluorescent protein GFP polC gfp was placed in single copy in the B subtilis chromosome under control of the endogenous polC promoter 5 PolCGFP supported DNA replication and cell growth when it was present as the only source of the catalytic subunit and it was visible as discrete fluorescent foci at or near midcell in most cells during exponential growth Fig 1A 6 In these cells the DNA occupied most of the cytoplasmic space and appeared to extend to the cell boundaries Fig 1B Two experimental observations indicated that the foci correspond to DNA polymerase at replication forks i the presence of foci was dependent on continued DNA synthesis and ii the number of foci per cell increased at faster growth rates We prevented reinitiation of DNA replication by inhibiting expression of DnaA which binds to oriC and is required for assembly of the replication complex 3 4 We fused dnaA to the LacIrepressible isopropyl D thiogalactopyranoside IPTG inducible promoter Pspac 7 so that transcription of dnaA was IPTG dependent In the presence of IPTG expression of dnaA 5 of the cells lacked the visible Table 1 The number of PolC GFP foci per cell