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Repair of DNA Containing Small Heterologous Sequences by Escherichia Coli: a Dissertation University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 1991-11-01 Repair of DNA Containing Small Heterologous Sequences by Escherichia Coli: a Dissertation Breck Olland Parker University of Massachusetts Medical School Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_diss Part of the Amino Acids, Peptides, and Proteins Commons, Bacteria Commons, and the Genetic Phenomena Commons Repository Citation Parker BO. (1991). Repair of DNA Containing Small Heterologous Sequences by Escherichia Coli: a Dissertation. GSBS Dissertations and Theses. https://doi.org/10.13028/9f9m-8d07. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/123 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. Repair of DNA Containing Small Heterologous Sequences by Escherichia coli. A Dissertation Presented Breck Olland Parker Submitted to the Faculty of the Graduate School of Biomedical Sciences at the University of Massachusetts Medcal School in parial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY IN MEDICAL SCIENCES PHARMACOLOGY November 1991 Repair of DNA Containing Small Heterologous Sequences by Escherichia coli. A Dissertation By Breck Olland Parker Approved as to style and content by: Larry W. Hardy Chairman of Committee Eric von Hofe Member of Committee Michael R. Volkert Member of Committee Kendall L. Knight Member of Committee Bruce F. Demple Member of Committee Martin G. Marinus Thesis Advisor Thomas B. Miller, Jr. Dean of the Graduate School of Biomedical Sciences Department of Pharmacology November 1991 ii ACKNOWLEDGEMENTS I want to extend my sincere grtitude to my thesis advisor, Marn Marnus, for guiding me through the rigors of gr duate school. I'm especially grateful for his patience and encouragement when times were rough, and for sharng in my excitement and enthu- siasm when things went well. His guidance and frendship over the last five years is greatly appreciated. I am also very grateful to the members of my thesis advisory commttee: Mike Vol- kert, Bob Lahue and Lar Hardy, for their constat encouragement, invaluable advice and infectious enthusiasm for science. Thans Lar, for always providing that different point of view, for always asking the one question that keeps me humble, and for makng me work harder for fear that you ll continue to ask such questions. I extend my gratitude to Neal Brown and the rest of the Pharacology deparment faculty for their support and guidance over the last five years. A special thanks to our deparmental statistician, Bob Lew, for those early morning and weekend philosophical interludes while waiting for the coffee to brew. Finally, my deepest gratitude goes to my famiy. Their steadfast support and encouragement have helped to make this achievement possible. I especially want to than my wife, Linda, who has been my parer in everything over the last seven years, for her love, support and patience, my son, Andrew, who has been a constant source of inspiration and motivation, and my Mom and Dad, who have been a stabilzing influence throughout my life and have always supportd and encouraged me regardless of what I chose to do. S. Thans Fran, for constantly reminding me that there is life outside of science, i.e. Walter s opprobrious legend. Thans for the Bud. For his love of life, love of famly and love of God, I dedcate this thesis to Roland C. Hallen. (Aug. 16, 1907 to July 1 , 1983) Prostate Cancer REPAIR OF DNA CONTAINING SMALL HETEROLOGOUS SEQUENCES BY Escherichia coli. November 1991 Breck Olland Parker B.S., . University of Massachusetts, Amherst Ph.D., Graduate School of Biomedical Sciences at the University of Massachusetts Medcal School Directed by Marn G. Marnus ABSTRACT The Dam-dependent mismatch repai system of Escherichia coli is par of a large network of DNA sureilance and error avoidance systems that identify and repai DNA damage. In this thesis, I have investigated the Dam-dependent mismatch repai system of E. coli and its role in the recognition and repai of DNA substrte molecules containing small inserton/deletion heterologies. This investigation was divided into two pars: the first par utilzed genetic techniques to evaluate the specifcity of repai and the second par utilized biochemical approaches into the recognition of inserton/deletion heterolo- gies. I have developed a sensitive in vivo transformation system to rapidly evaluate the repai of small inserton/deletion heterologies by Dam-dependent mismatch repai. Het- eroduplexes were constrcted, for each state of methylation of d(GA TC) sequences, by anealing single strand DNA to the linearzed complementa strand of duplex DNA. The unmethylated single strand DNA was isolated from f1 phage (R408) propagated on a strain of E. coli contaning the dam- 16 alele (Chapter 2) to eliminate the possibilty of residual Dam-methylation of d(GA TC) sequences. Tranformation of E. coli indicator strains with heteroduplexes containing 1 4 and 5 base inserton/deletion heterolo- gies were scored for repair based on colony color. The results of these experiments show that the Dam-dependent mismatch repai system can recognize and repai 1, 2 and 3 base heterologies as well as repaing Gff mispais (Chapters 3 and 4). The repai of 4 base heterologies was marginal, while no repai was observed with 5 base heterologies (Chap- ters 3 and 4). Repai of the 1 3 and 4 base heterologies proceeded in a Dam- dependent process that required the gene products of mutL, mutS, and I have demonstrated that MutS protein from both Salmonella typhimurium and coli can recognize and bind in vitro to the same 1, 3 and 4 base heterologies used for the genetic studies above (Chapters 4 and 5). In fact, MutS protein binds to 1, 2 and 3 base heterologies with greater affinity than it binds to a Grr mismatch. The in vitro observation that MutS does not bind to 5 base heterologies is consistent with the in vivo observation that 5 base heterologies are not subject to repair. I have also shown that MutS protein specifically binds to 1, 2 and 3 base heterologies since MutS protects about 25 base pais of DNA flanng the site of the heterology from DNaseI digestion. The results of the genetic and biochemical experients describe in this thesis (and summarzed above) serve to re-emphasize the importance of the role that methyl-diected mismatch repai plays in mutation avoidance, and hence in the preservation of genetic integrty. """"""""""" ...... .......... .................... .............................................. .......................... ............ .......... .......................... ............................... ...........,... TABLE OF CONTENTS Title Page....... ................................................ .......... Signature Page................................... ........ .............. Acknowledgements....................................... .................................,............................. iii Dedication..................................................................................................................... iv Abstract... Table of Contents......... .............................................................. .......... ......................... vii List of Tables...... .................................................................. .........................."............ List of Figues................................................................... ............................................ Xlll CHAPTER 1: Introduction.......... .............. .............. .............................................. A. The conservation of genetic informtion........................................................... 2 Systems involved in repaing DNA as a result of endogenous damage........... Systems that protect against environmenta and chemical insult...................... Initial misincorporation of deoxynucleotides is dependent on base.................. selection by DNA polymerase il holoenzyme and the gene product of mutT. The 3' -5' exonuc1eolytic proofreading function of DNA polymerase II.......... 7 Dam-dependent mismatch repai corrects errors after passage of the................ 9 replication fork. B. d(GA TC) Sequences and Dam-dependent Mismatch Repair............................. DNA adenine methyltransferase (Dam) is encoded by the dam gene................ 11 vii Dam-methylation of adenine in d(GATC) sequences diects the....................... strandedess of repai. Dam-dependent mismatch correction requires at least one d(GA TC)............... site for efficient correction of a mismatch. C. Specificity of Dam-dependent Mismatch Repai............................................. Trasition mispais are corrected more efficiently than transversion............... mispais. Repai of small frame shift mutations................................................................ D. The Biochemistr of Dam-dependent Mismatch Repair.................................. Isolation and characterization of MutS.............................................................. 21 Isolation and characterization of MutH............................................................. Isolation and characterization of MutL.............................................................. Components of excision
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