Blocking rolling circle replication with a UV lesion creates a deletion hotspot.
UV light irradiation increases genetic instability by causing mutations and deletions. The mechanism of UV-induced rearrangements was investigated making use of deletion-prone plasmids. Chimeric plasmids carrying pBR322 and M13 replication origins undergo deletions that join the M13 replication origin to a random nucleotide. A restriction fragment was UV irradiated, introduced into such a hybrid plasmid and deletions formed at the M13 origin were analysed. In most of the deletant molecules, the M13 replication nick site was linked to a nucleotide in the irradiated fragment, showing that UV lesions are deletion hotspots. These deletions were independent of the UvrABC excision repair proteins, suggesting that the deletogenic structure is the lesion itself and not a repair intermediate. They were not found in the absence of M13 replication, indicating that they result from the encounter of the M13 replication fork with the UV lesion. Furthermore, UV-induced deletions occurred independently of pBR322 replication. We conclude that, in contrast to pBR322 replication forks, M13 replication forks blocked by UV lesions are deletion prone. We propose that the deletion-prone properties of a UV-arrested polymerase depend on the associated helicase.
Seigneur
M
M
Institut National de la Recherche Agronomique, Jouy en Josas, France.
Ehrlich
S D
SD
Michel
B
B
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D017104:Bacteriophage M13 / Q000235:genetics; D001483:Base Sequence; D004261:DNA Replication / Q000528:radiation effects*; D004279:DNA, Viral / Q000096:biosynthesis* / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004706:Endodeoxyribonucleases; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D010957:Plasmids; D018741:Replication Origin / Q000528:radiation effects; D017384:Sequence Deletion; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D004706:Endodeoxyribonucleases; D015246:Deoxyribonuclease EcoRI; C045874:endodeoxyribonuclease StyI; D015252:Deoxyribonucleases, Type II Site-Specific; C066567:UvrC protein, E coli; C068631:UvrA protein, E coli; D000251:Adenosine Triphosphatases