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DNA structure(s) recognized and bound by large subunit of Replication Factor C (ls RFC) in Drosophila melanogaster
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Alternative (non-B type) DNA structures are thought to be formed as intermediates in developmentally important mechanisms such as DNA replication, DNA repair, and genetic recombination. It is proposed that these alternative DNA structure(s) and the proteins that recognize them may play an important role in the regulation of cellular mechanisms. There has been an enhancement in the knowledge of proteins that bind to the DNA by recognizing specific sequence and/or structural aspects of the DNA. lsRFC (large subunit Replication Factor C) is a protein that binds to the DNA in a sequence independent manner and recognizes structural determinant(s) in the DNA structure. lsRFC is an essential part of the five subunit RFC complex involved in the process of eukaryotic DNA replication. RFC complex loads PCNA and DNA polymerase delta onto the replication template during replication and is required both for leading and lagging strand synthesis. Southwestern analyses using synthetic oligonucleotides containing internal complementary sequences to form DNA structures reveal that structural determinants present in the branched DNA structures are most strongly bound by lsRFC. Osmium tetroxide chemical modification of these synthetic DNA structures verifies the formation of the predicted branched structures. Tsurimoto and Stillman (I 99 1) used DNase and micrococcal nuclease footprinting assays to define a primer template junction as a substrate for the RFC complex binding. A synthetic primer-template junction is bound by lsRFC in nitrocellulose filter binding (Southwestern) assays; however, it is bound approximately five fold less efficiently than branched DNA structures but better than single stranded DNA or duplex DNA. This suggests that HP96 contains a determinant that is preferentially recognized by lsRFC. To investigate the determinant present, chemical probing using osmium tetroxide was performed and reveals either single strand or distorted duplex nature at the proposed primer template junction. Based on the Southwestern assays and the chemical modification data, it is proposed that lsRFC preferentially recognizes and binds angles or bends in the DNA and may recognize the replication fork formed at the junction between parental and daughter strands. This proposed intimate association of lsRFC with the replication fork may influence the progression of the fork.
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Gaur, Lalit Kumar (1995). DNA structure(s) recognized and bound by large subunit of Replication Factor C (ls RFC) in Drosophila melanogaster. Master's thesis, Texas A&M University. Available electronically from
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