Paulsson:Replication inhibitors

From OpenWetWare

Jump to: navigation, search

Home        Group Meeting        Protocols        Inventory        Journal Watch        Top 100        Links        Facts of Life       


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


Contents

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)

Personal tools