COMPOSITE PATTERNS IN NEUTRAL NEUTRAL 2-DIMENSIONAL GELS DEMONSTRATE INEFFICIENT REPLICATION ORIGIN USAGE/

The neutral/neutral two-dimensional (2-D) gel replicon mapping techniq ue has been used to great advantage to localize and characterize origi ns of replication. Interestingly, many yeast origins display a composi te pattern consisting of both a bubble are and a single-fork are Moreo ver, in every instance in which neutral/neutral 2-D gels have been use d to analyze origins in higher eukaryotic cells, two or more adjacent fragments display these composite patterns. We believe that composite patterns signal inefficient origin usage in yeast cells because the re plicators in question are not active in every cell cycle and in higher eukaryotic replicons because initiation sites are chosen from among m any potential sites lying within a zone. However, others have suggeste d that the single-fork arcs in these composite gel patterns arise from nicking activity that converts replication bubbles to branched struct ures that comigrate with bona fide single forks. Here, we have used th ree different replicon mapping strategies to show that broken simian v irus 40 replication bubbles trace unique arcs that are clearly disting uishable from classic, intact single forks. Thus, it is likely that co mposite 2-D gel patterns represent origins that are inefficiently util ized.