On the differential DNA secondary sructure mutagenesis in the leading and lagging strands during DNA replication in Escherichia coli /
Cellular DNA replication has evolved to accurately reproduce B-form DNA, while allowing mutations to occur at a low level. The accuracy of replication can be altered by defined order sequence DNA (dosDNA). dosDNA elements may form secondary structures, and these structures can have a profound effect...
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
1998.
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| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=737704531&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | Cellular DNA replication has evolved to accurately reproduce B-form DNA, while allowing mutations to occur at a low level. The accuracy of replication can be altered by defined order sequence DNA (dosDNA). dosDNA elements may form secondary structures, and these structures can have a profound effect on mutagenesis. The concurrent model of DNA replication in E. coli suggests a dimeric DNA polymerase III complex that simultaneously synthesizes both the leading and the lagging strands. While the leading-strand polymerase is processive, the lagging-strand polymerase must synthesis DNA discontinuously and temporally later than synthesis of the complementary sequences of the other strand. This dimeric nature of the polymerase has potential for strand specific mutagenic events. This dissertation explores mutational systems to explain how DNA structure differentially influences mutagenesis in the leading and lagging strands.In the first system, the frequencies of deletion of short sequences inserted into the chloramphenicol acetyltransferase (CAT) gene were measured in pBR325. Deletion of these mutation inserts may be mediated by slipped misalignment during DNA replication. These sequences contained an inverted repeat that was asymmetric with respect to direct repeats. Different misaligned structural intermediates could form in the leading and lagging strands dependent upon orientation of the insert and/or orientation of the CAT gene,and influence the mutation frequency. These differential structures showed that when a hairpin can form in the lagging strand mutagenesis is increased. In the second system, a quasipalindrome was used to study frameshift mutagenesis. Plasmid pJT7 is a pBR325 derivative that contains a -I frameshift mutation in the CAT gene. This -I frameshift creates a 17 hp quasipalindrome within the gene, and the Cm' phenotype can be restored by a + I frameshi ft of the quasipalindrome to a perfect palindrome. A genetic selection to demonstrate that this leading strand preferential spontaneous mutation can occur by an intermolecular strand switch was developed. We find that the mutation occurs by a mechanism that is preferential for the leading strand during DNA replication. Both of the systems studied in this dissertation demonstrate the importance of DNA secondary structure to mutagenesis. |
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| Item Description: | Vita. "Major Subject: Biochemistry". |
| Physical Description: | xii, 131 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilms Inc. |
| Bibliography: | Includes bibliographical references: pages 121-128. |