Paulsson:Replication inhibitors
What I want: fast (cell permeant) plasmid DNA replication inhibitor that doesn't interfere with transcription or protein synthesis
Possibilities: drugs, operator arrays, trx through ori, ts-alleles, combinations
Classes of drugs (generally defined by cellular target)
Gyrase inhibitors
Good review in Biochemistry 27(7):2253, Karl Drlica and Robert Franco, 1988.
gyrA
quinolones (naladixic acid, oxolinic acid, ciprofloxacin)
MOA: create gyrase-DNA adduct that can be processed into strand breaks by protein denaturation.
gyrB
coumarins (novobiocin, coumermycin A1)
The good: fast and full inhibition
The bad: also interferes with RNA polymerase, affects transcription of different genes to varying degree
Polymerase inhibitors
nucleotide analogs - generally not permeant
cells can be made permeable with toluene and other treatments
[1]
Helicase (eg. DnaB, RepA of pRSF1010) inhibitors
Flavones [2]
DNA intercalators
ethidium
acridine
Nucleotide reductase inhibitors
Hydroxyurea (HU)(use at 50mM, make fresh 1M stock in water or media; some store at -20deg as 1M stock)
The good: 1)fast inhibition; 2) limited effect on RNA synthesis, protein synthesis, cell viability; 3) reversible(?); 4) better than ts-allele of nrdA [3], [4]
The bad: 1) in Bacillus HU has preferential effect on chromosomal DNA replication, very little effect on plasmid replication
[5]
2) also preferential effect on chromosome replication found in Proteus mirabilis (Mol.Gen.Genet. 138:281, 75)
thermosensitive alleles
dnaA
large collection of well characterized alleles, but R1 replication is DnaA independent! and different ori's have different DnaA dependencies
pol###
nrdA101
interesting allele, but not good for my purposes (see HU ref, 2007)
