Rolling-circle replication of UV-irradiated duplex DNA in the phi X174 replicative-form----single-strand replication system in vitro
Cloning of the phi X174 viral origin of replication into phage M13mp8 produced an M13-phi X174 chimera, the DNA of which directed efficient replicative-form----single-strand rolling-circle replication in vitro. This replication assay was performed with purified phi X174-encoded gene A protein, Escherichia coli rep helicase, single-stranded DNA-binding protein, and DNA polymerase III holoenzyme. The nicking of replicative-form I (RFI) DNA by gene A protein was essentially unaffected by the presence of UV lesions in the DNA. However, unwinding of UV-irradiated DNA by the rep helicase was inhibited twofold as compared with unwinding of the unirradiated substrate. UV irradiation of the substrate DNA caused a strong inhibition in its ability to direct DNA synthesis. However, even DNA preparations that contained as many as 10 photodimers per molecule still supported the synthesis of progeny full-length single-stranded DNA. The appearance of full-length radiolabeled products implied at least two full rounds of replication, since the first round released the unlabeled plus viral strand of the duplex DNA. Pretreatment of the UV-irradiated DNA substrate with purified pyrimidine dimer endonuclease from Micrococcus luteus, which converted photodimer-containing supercoiled RFI DNA into relaxed, nicked RFII DNA and thus prevented its replication, reduced DNA synthesis by 70%. Analysis of radiolabeled replication products by agarose gel electrophoresis followed by autoradiography revealed that this decrease was due to a reduction in the synthesis of progeny full-length single-stranded DNA. This implies that 70 to 80% of the full-length DNA products produced in this system were synthesized on molecules that carried photodimers.
- Research Organization:
- Weizmann Inst. of Science, Rehovot (Israel)
- OSTI ID:
- 5716381
- Journal Information:
- J. Bacteriol.; (United States), Vol. 171:6
- Country of Publication:
- United States
- Language:
- English
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59 BASIC BIOLOGICAL SCIENCES
BACTERIOPHAGES
DNA REPLICATION
DNA
GENETIC RADIATION EFFECTS
ATP-ASE
AUTORADIOGRAPHY
DNA-CLONING
ELECTROPHORESIS
IN VITRO
INHIBITION
PROTEINS
PYRIMIDINE DIMERS
ULTRAVIOLET RADIATION
ACID ANHYDRASES
BIOLOGICAL EFFECTS
BIOLOGICAL RADIATION EFFECTS
CLONING
DNA HYBRIDIZATION
ELECTROMAGNETIC RADIATION
ENZYMES
GENETIC EFFECTS
HYBRIDIZATION
HYDROLASES
MICROORGANISMS
NUCLEIC ACID REPLICATION
NUCLEIC ACIDS
ORGANIC COMPOUNDS
PARASITES
PHOSPHOHYDROLASES
RADIATION EFFECTS
RADIATIONS
VIRUSES
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