Differential replication of a single, UV-induced lesion in the leading or lagging strand by a human cell extract: fork uncoupling or gap formation.
We have constructed simian virus 40 minireplicons containing uniquely placed cis,syn-thymine dimers (T <> T) for the analysis of leading- and lagging-strand bypass replication. Assaying for replication in a human cell-free extract through the analysis of full-size labeled product molecules and restriction fragments spanning the T <> T site resulted in the following findings: (i) The primary site of synthesis blockage with T <> T in either the leading or lagging strand was one nucleotide before the lesion. (ii) Replicative bypass of T <> T was detected in both leading and lagging strands. The efficiency of synthesis past T <> T was 22% for leading-strand T <> T and 13% for lagging-strand T <> T. (iii) The lagging-strand T <> T resulted in blocked retrograde synthesis with the replication fork proceeding past the lesion, resulting in daughter molecules containing small gaps (form II' DNA). (iv) With T <> T in the leading-strand template, both the leading and lagging strands were blocked, representing a stalled replication fork. Uncoupling of the concerted synthesis of the two strands of the replication fork was observed, resulting in preferential elongation of the undamaged lagging strand. These data support a model of selective reinitiation downstream from the lesion on lagging strands due to Okazaki synthesis, with no reinitiation close to the damage site on leading strands [Meneghini, R. & Hanawalt, P.C. (1976) Biochim. Biophys. Acta 425, 428-437].
Svoboda
D L
DL
Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill 27599-7295, USA.
Vos
J M
JM
D001483:Base Sequence; D004249:DNA Damage; D017931:DNA Primers; D004261:DNA Replication; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D011740:Pyrimidine Dimers; D012093:Replicon; D015183:Restriction Mapping; D013539:Simian virus 40 / Q000235:genetics / Q000502:physiology; D013698:Templates, Genetic; D014020:Tissue Extracts / Q000502:physiology; D014466:Ultraviolet Rays; D014779:Virus Replication
D017931:DNA Primers; D011740:Pyrimidine Dimers; D014020:Tissue Extracts