Intrinsic DNA structures and their role in DNA replication
Abstract
Sequence-dependent structures involving DNA bending were initially studied by computer analysis. Modelling of synthetic oligonucleotides and of 107 kb of natural sequences have results which predicted structures that were consistent with published electrophoretic data. The analysis was therefore extended to the study of DNA structures involved in chromosome maintenance. Centromeric DNAs from yeast were found to have sequences in their functional elements which caused them to adopt an unusually straight structure. Sequences capable of conferring autonomous replicating ability on plasmids in yeast were found to have two structural domains, one consisting of unusually straight sequences and other of bent DNA. A novel two-dimensional gel electrophoretic method was then used to examine the structural domains in replicators isolated from four different plant genomes and from the Ti plasmid. Bent DNA was found to be associated with the ARS elements, and the ARS consensus sequence was flanked by unusually straight structures. The results show that the ARS elements from genomes of higher plants have structural and sequence features in common with ARS elements from yeast and higher animals. The higher-order DNA structure of a variety of origins of replication from plasmids, phages, and bacteria was also examined. The results demonstrate that the structural features of eukaryotic replicators are found in prokaryotes, providing new insight into the universal role of conserved DNA structures in DNA replication. Finally, the unusually straight and bending domains were cloned from synthetic oligomers and introduced into yeast in an attempt to produce an artificial origin of replication. The inability of these constructs to support replication suggests that our understanding of the requirements for a complete replicator is incomplete.
Degree
Ph.D.
Advisors
Anderson, Purdue University.
Subject Area
Molecular biology
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