REPAIR IN Escherichia coli OF A PSORALEN‐DNA INTERSTRAND CROSSLINK SITE SPECIFICALLY INTRODUCED INTO T₄₁₀A₄₁₁ OF THE PLASMID pUC 19

Abstract

Abstract— Escherichia coli was transformed with a pUC 19 plasmid which contained a 4,5′,8‐trimethylpsoralen cross‐link specifically at position T410‐A411, and transformants in which the cross‐link had been repaired were scored on the basis of ampicillin resistance. Repair was only seen after induction of the SOS‐system by shortwave ultraviolet irradiation. The repair efficiency was 6%, and of 100 colonies analyzed only two was found to be mutated at the cross‐linked site: a T‐A transversion was observed at position 410. A repair mechanism which does not involve recombination is proposed.

Nucleotide excision repair in Escherichia coli.

“…This strain was found to overproduce a 55-kdal protein. On the basis of survival data, Zhen et al (284) have proposed a novel glycosylase activity for the repair of cross-linked psoralen adducts. Sladek et al have isolated a novel enzymatic activity from uvr E. coli that acts only on psoralen monoadducts, leads to incisions of supercoiled DNA, is Mg2+ independent, and leads to the release of labeled psoralen (F. M. Sladek, B. Dynlacht, and P. Howard-Flanders, J.…”

Mutagenesis induced by site specifically placed 4'-hydroxymethyl-4,5',8-trimethylpsoralen adducts

“…The mutations are targeted and consist of transversions or single nucleotide deletions when the modified plasmid is introduced into bacterial or mamalian cells . A pUC19 plasmid containing a site-specifically placed 4,5',8-trimethylpsoralen crosslink has been used to transform E. cofi (38). Only two mutants were sequenced and each consisted of a A:T to T:A transversion.…”

Incision by UvrABC excinuclease is a step in the path to mutagenesis by psoralen crosslinks in Escherichia coli.

“…The mechanism by which psoralen crosslinks produce mutations in E. coli is not known but it appears to require SOS functions (9,12,13). These are a set ofdamage-inducible cellular responses that lead to increased DNA repair and to mutagenesis by a variety of DNA-damaging agents (14)(15)(16).…”

“…However, it was only recently that crosslinks alone were proven to be mutagenic and not just lethal. Zhen et al (9) constructed a plasmid free of monoadducts which contained a single psoralen crosslink in a given position. Upon transformation into excisionproficient (Uvr+) E. coli cells, the crosslinked plasmid yielded mutations at a relatively high frequency (3%).…”