DNA Replication in Bacillus subtilis

Notes: 5-bromouracil incorporation into DNA

-Pyrimidine analog. Bromine is substituted for methyl at C5. The van der waals radius is maintained.
- Sensitizes the DNA by 10x to Ultra Violet Radiation Mutsumoto K. et al. 1974.
- Mutagenic and lethal at some doses. Mutsumoto K. et al. 1974.
- 5-bromouracil exists as a tautomer. It is thought to pair with A then rearrange and upon replication pair with G causing A:T to G:C mutations. Diagrams.
- Sporulating cells with BU DNA cannot complete spore formation in the hands of ADAMS and WAKE in 1980
Does this imply that there is something essential which is different about DNA translocation in vegetative vs. sporulating cells?


Notes: HPUra incorporation into DNA

-Binds to cytosine
-Inhibits polymerase III of B. subtilis. It does not inhibit repair synthesis or synthesis directed by phages (E. coli pol I, B. sub pol I or polymerase from T4). (Gass, Low and Cozarelli 1973)
- Dithioreitol (a reducing agent) can activate the drug. Activation of this sort is required for this function. (Gass, Low and Cozarelli 1973)
-The Effect is competitively excluded by high concentrations of dGTP. So the template is likely involved but the action is thought to be on the polymerase enzyme itself (because of the specificity). (Gass, Low and Cozarelli 1973)


Notes: [3H] Thymine incorporation into DNA

-Thymidine is rapidly converted to Thymine in thymine requiring spores of Bacillus (cited in Callister H., Le Mesurier S., Wake R.G.)
-1.5 ug/ml used in the Bacillus subtilis studies of WAKE
- "3H- Thymidine is a defective tool with which to measure rates of DNA synthesis" FASEB. The authors are looking at the rates of other nucleotides incorporation in [3H] thymidine experimenits *in eukaryotic cells* and examining a dose dependent decline in cell survival. >= 1 mci/ml is cytotozic to these cells (also used comet assay)Hu et al. 20002
-Changing concentrations of radiolabeled Thymine (10 u ci/10 ml culture) does not appear to affect cell survival of Bacillus megaterium by environmental microbiologists in AEM 1992.

Notes on use of Mutation DnaB134

-This mutation is the standard mutation used to make initiation of replication in B. subtilis temperature dependent. 45C is non-permissive, 32-34 is permissive (see RNA polymerase drives DNA replication for an example of Dworkin and Losick using exactly the same mutation in their studies).
-DnaB is thought to be involved in primosome assembly (mentioned in Grossman 2000).

PAPERS:


1977
Initiation of Deoxyribonucleic Acid Replication in Germinating Spores of Bacillus subtilis 168 Carrying the dnaB(Ts)134 Mutation (Callister H., Le Mesurier S., Wake R.G.)

DNA synthesis occurs in spores of the TsB134 strain germinating at 45C. A normal round of replication can occur in some of these germinating spores and this is dependent on the action of the dnaB134 gene product. 23% to 58% of a round of replication occures after 240 minutes at 45C as assayed by 5BU plus thymine incorporation (ts has not reverted).
Is this repair or replication?
The early replicated DNA appears to include the MetB5 and PurA16 markers by density gradient analyis.
*note, in order to eliminate the possibility that repair synthesis is taking place they look at the markers in their HH, LH and LL species compared to the radioactivity incorporation and state that radioactivity is associated exclusively with LH (replicated) species and therefore there is no repair synthesis evident. This is crap though because if repair synthesis was taking place in every cell and leading to incorporation of radio label EVERYWHERE this would simply be counted as noise and the replication at the ORIGIN in EVERY CELL is clearly going to overwhelm such a signal. There could easily be lots of repair going on in these cells.

They went on to anaylze the effect of small changes in temperature on the DNA synthesis that does occur. etc etc.

1980

"Highly Specific Labeling of the Bacillus subtilis Chromosome Terminus" Adams and Wake 1980

NOTE: This paper is relying on HPUra inactivation of replication and it is notable that this does not stop replication that occurs by polymerases other than POL III (see Cozarelli).

The experiments described were initially going to be performed using BU to label the DNA but BU DNA was never found in the spores....

[3H]-Thymine was used instead.

If replication was stopped by the addition of HPura at 60 minutes only 20% of the spore yield was obtained. Therefore Labeled Thymine was added at 60 minutes and replication was stopped by the addition of HPura at 70 minutes.
Spore formation was allowed to proceed (20 hours) and the spores that did manage to form were examined. Chromosomal location of the label was determined by germinating these spores in the presence of BU (60 minutes into germination) and performing Density transfer experiments at 100, 150 and 200 minutes.

As time passed more of the DNA was replicated as evidenced by incorporation of the BU into the DNA.
Conclusion: Spores inlcude only complete chromosomes.
NOTE: Density matters for details of density gradient experimental method (PNAS)


Paper:
"Analysis of the terminus region of the Bacillus subtilis chromosome" Weiss, A.S., Hariharan I.K. and Wake R. G. 1981

Essentially the same experiment was run initially with the spores that formed in the TS strain with the addition of more DNA by resuspending the cells. A digestion was performed using EcoRI BamHI and SalI. THis results in a set of discrete bands that are labeled although their ordering can not be determined and the genome sequence was not known at the time.
A finer scale set of experiments are done with smaller time points and only SalI labelling but the order is not known. These experiments put the terminus in a region between 2% or .4% of the total genome.

Paper:
"Restriction map of DNA spanning the replication terminus of the Bacillus subtilis chromosome"
Weiss and Wake 1983
in progress... This is really the critical paper and it would be really great to be able to look at the restriction digests while doing this...