Analysis of single molecules by EM provides a powerful approach to study the mechanics of DNA replication. The continuing focus in this renewal application is how eukaryotic origins are opened by proteins and how DNA strands are looped at the fork to coordinate leading and lagging strand replication. A statistically large number of molecules are examined and this information is combined with data from biochemical assays. A highly interactive program has been established with Dr. Steve Bell (yeast ORC), Drs. Tom Broker and Louise Chow (human papillomaviruses), Dr. Charles Richardson (T7 replication), Dr. Nancy Nossal (T4 replication), and Dr. Charles McHenry (E. coli). The structure of the yeast Origin Recognition Complex will be examined, both alone and assembled into a pre-RC with Mcm and Cdc6 factors using a variety of EM methods. A lac repressor affinity trap will be utilized to isolate replicating minichromosomes containing the ARS 1 from S. cerevisiae cells and the structure and architecture of the origin complexes examined. The structure Of the HPV origin bound by E1, E2 and host proteins will be examined using a gentle EM method developed in the past renewal (glycerol spray/low voltage EM). An affinity trap will be placed in plasmids containing the HPV origin. These plasmids will be allowed to replicate in human cell extracts and then gently isolated and examined by EM. The structure of moving forks will continue to be investigated in the T7 and T4 systems. Novel nano-scale biopointers developed in the last renewal will be used to determine the number of T4 and T7 DNA polymerase molecules present in the replication complex. Replication complexes assembled on a 400 bp mini-circle template will be examined to obtain a higher resolution structure of the replisomes. The structure and role of the single strand DNA-SSB bobbins will be examined as they may provide the central 'gears' of the replication machine at the fork. These studies will be extended to the more complex E. coli system to provide a structural understanding of the E. coli replisome and location of the individual protein components.
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