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Title: Studies on the genetic and biochemical properties of a PUVA hyper-resistant mutant of Escherichia coli
Author: Holland, Julie
ISNI:       0000 0001 3580 4154
Awarding Body: Nottingham Polytechnic
Current Institution: Nottingham Trent University
Date of Award: 1990
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8-methoxypsoralen plus near ultraviolet light (PUVA) is used to treat psoriasis and other cutaneous skin disorders. Much of the lethal and theraputic effects of PUVA treatment are attributed to the drug's ability to covalently bind to DNA, forming crosslinks and thereby preventing its replication and transcription. Many organisms including bacteria have developed mechanisms to repair DNA crosslinking damage induced by PUVA. A mutant of Escherichia coli (SA270), hyper-resistant to PUVA, was shown to have simultaneously become hyper-resistant to the DNA crosslinking agents mitomycin C and nitrogen mustard,but not to monofunctional DNA damaging agents such as far ultraviolet light. SDS-Polyacrylamide gel electrophoresis of total protein extract showed that a 55kd protein was over-expressed in strain SA270, but not in its wild type parent strain KL16. A mutation (designated puvR), located at 57.2 minutes on the E.coli chromosome, concomitantly caused over-expression of the 55kd protein and PUVA hyper-resistance. Studies with radiolabelled 8-methoxypsoralen showed that the PUVA hyper-resistant phenotype of SA270 was not due to a decreased permeability barrier. It has been previously suggested that the PUVA hyper-resistant phenotype of SA270 was due to an enhanced DNA repair mechanism involving recA; however, in an experiment showing induction of the RecA protein, no induction of the 55kd protein was detected. Therefore the recA gene was not directly involved in controlling expression of the 55kd protein. However, complementation studies showed that the 55kd protein was negatively regulated and may therefore be indirectly induced by DNA crosslinks. Evidence also suggested that the recF gene product may be involved in regulating expression of the 55kd protein. A number of DNA repair-deficient mutants of E.coli (recA, uvrA, uvrC, recBC, reef and recN) were all sensitive to PUVA. This implicated the UvrABC excision repair pathway and the RecBC and RecF post-replication recombination repair pathways in DNA crosslink repair. Lambda DNA damaged with PUVA was degraded by total protein extract of strain SA270 with greater efficiency than with total protein extract of KL16. This phenomenon was dependent on the presence of the 55kd protein since total protein extract of a PUVA hyper-sensitive mutant did not degrade X DNA. In contrast, DNA incubated with purified 55kd protein was not degraded, suggesting that the protein was dependent on other proteins for its activity. Since the uvrC mutant derivative of SA270 was not hypersensitive to PUVA, the 55kd protein may form part of the UvrAB complex, thereby rendering its endonuclease activity specific for DNA crosslinks. Alternatively, there may exist a DNA repair pathway specific for crosslinked DNA damage which involves the 55kd protein in as yet an unknown role.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Furocoumarins[Psoralens]