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. Thus, similarly to its activity on UV-irradiated single-stranded DNA, DNA polymerase III holenzyme can bypass pyrimidine photodimers in the more complex replicative form --->single-strand replication, which involves, in addition to the polymerizing activity, the unwinding of the duplex by the rep helicase and the participation of a more complex multiprotein replisome.
Shavitt
O
O
Department of Biochemistry, Weizmann Institute of Science, Rehovot, Israel.
Livneh
Z
Z
D000251:Adenosine Triphosphatases / Q000502:physiology; D010584:Bacteriophage phi X 174 / Q000235:genetics*; D003001:Cloning, Molecular; D004249:DNA Damage; D004265:DNA Helicases; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000235:genetics*; D004279:DNA, Viral / Q000528:radiation effects*; D004268:DNA-Binding Proteins / Q000502:physiology; D029968:Escherichia coli Proteins; D066298:In Vitro Techniques; D011740:Pyrimidine Dimers; D012045:Regulatory Sequences, Nucleic Acid; D014466:Ultraviolet Rays; D014779:Virus Replication
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D011740:Pyrimidine Dimers; C506744:rep protein, E coli; D000251:Adenosine Triphosphatases; D004265:DNA Helicases