Abstract
DNA replication in eucaryotic cells is a complex process involving a variety of proteins that synthesize the leading and lagging strand in an asymmetric, coordinated manner. To investigate the effect of this asymmetry on the translesion synthesis of bulky lesions, we have constructed SV40 origin-containing plasmids with site-specific N-2-acetylaminofluorene adduct on either leading or lagging strand templates. These plasmids have been incubated with DNA replication-competent extracts made from human HeLa cells. Two-dimensional agarose gel electrophoresis analyses reveal a strong blockage of fork progression only when the N-2-acetylaminofluorene adduct is located on the leading strand template. Morever, the analysis revealed that replication with HeLa cell extracts of SV40 origin-dependent plasmids functions in both directions from the origin with equal efficiency but, probably due to an important asynchrony at the formation of the two forks, proceeds unidirectionally for a large number of individual molecules. The validity of the in vitro replication approach to study the fidelity of both leading- and lagging strand synthesis is discussed with regard to these new data.
Highlights
DNA lesions are usually repaired in an error-free manner, which eliminates most of the consequences of a DNA-damaging treatment
Two-dimensional agarose gel electrophoresis analyses reveal a strong blockage of fork progression only when the N-2acetylaminofluorene adduct is located on the leading strand template
Reaction products were digested with StyI restriction enzyme, which cuts once at the origin (Fig. 1A), and analyzed by two-dimensional agarose gel electrophoresis (Fig. 2)
Summary
DNA lesions are usually repaired in an error-free manner, which eliminates most of the consequences of a DNA-damaging treatment. Recent studies using in vitro SV40 origin-dependent replication with HeLa cell extracts have been made to determine the accuracy of leading or lagging strand synthesis (6 –9). The conclusions that were drawn were based on at least two assumptions: first, that replication of SV40-based plasmids is bidirectional and synchronous from the SV40 origin under the conditions used for in vitro DNA replication; and second, that replication forks are hindered by lesions on the leading or lagging strand template. We have constructed plasmid molecules that contain a single N-2-acetylaminofluorene (AAF) in either the leading or lagging strand and used these DNAs as templates for replication in vitro in human HeLa cell extracts. We have used the same set of SV40 origin-dependent DNAs to analyze the inhibition of the replication fork by lesions on either the leading or the lagging strand template. The usefulness of the in vitro SV40based replication assay to study the fidelity of the two DNA strand synthesis is discussed on the basis of our findings
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