Division of Mycobacterial Research National Institute for Medical Research, Mill Hill London RecA expression and DNA damage in mycobacteria Nicola A Thomas June 1998 A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in the Faculty of Science, University College of London. ProQuest Number: U642358 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest U642358 Published by ProQuest LLC(2015). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 A bstr a ct Bacterial responses to DNA damage are highly conserved. One system, the SOS response, involves the co-ordinately induced expression of over 20 unlinked genes. The RecA protein plays a central role in the regulation of this response via its interaction with a repressor protein, LexA. The recA gene of Mycobacterium tuberculosis has previously been cloned and sequenced (Davis et al, 1991) and a LexA homologue has recently been identified (Movahedzadeh, 1996). The intracellular lifestyle of pathogenic mycobacteria constantly exposes them to hostile agents such as hydrogen peroxide. Consequently, the response of mycobacteria to DNA damage is of great interest. In this study the basal levels of RecA protein were shown to be elevated in M. microti and M. smegmatis compared to those found in M. tuberculosis, M. bovis BCG and a wide range of both saprophytic and pathogenic mycobacteria. Expression of this protein was shown to be inducible in both M. smegmatis and M tuberculosis in response to DNA damaging agents, although the kinetics of induction were quite different. A reduction in the levels of LexA following treatment of M. smegmatis, M. tuberculosis and M. microti with a DNA damaging agent, probably as a result of LexA cleavage, provided further corroboratory evidence to suggest that the essential regulatory elements of the SOS response may have been conserved. There was no evidence of RecA induction in M. microti. Sequencing of the M microti recA gene and 700bp of upstream sequence revealed a high degree of homology with equivalent sequences m M tuberculosis', including a putative LexA binding site. M. microti recA was shown to be inducible in M. smegmatis mc^l55 in response to DNA damage using a transcriptional fusion of the putative regulatory elements of this gene to the reporter gene chloramphenicol-acetyl transferase (CAT). Thus it appears that the kinetics of induction are controlled at the level of the host cell; this was further investigated by studying the interaction of purified mycobacterial LexA protein and M. microti cell-free extracts with the putative LexA binding site. T h e T e .\ï p t a t î o n o f S i A n t h o n y Israel van Meckenem ‘Sometimes it felt like this..,” A cknowledgements I wish to thank Dr. M. Joseph Colston and Dr. Elaine Davis for their invaluable input and guidance over the course of the last four years. They have consistently provided good advice surely no PhD. student could ask for more. I also wish to thank Patricia Brooks for her technical advice as well as Dr. Papavinasasundaram and Dr. Gili Bachrach for their interest and helpful discussions. A special thanks goes to Bob, Vangelis, Colin, Pat, Edith and Fran for their friendship, good humour and support over the past few years. Beyond academia, I owe a great debt to Mark who has made my existence far more pleasant in many ways. To my parents T a b l e o f c o n t e n t s T a b l e o f c o n t e n t s ............................................................................................................................................................................................ 5 L is t OF F i g u r e s ....................................................................................................................................................................................................10 L is t OF T a b l e s ..................................................................................................................................................................................................... 12 L ist o f A bbreviations .................................................................................................................................................................................13 1.0 I ntroduction ............................................................................................................................... 15 1.1 The Mycobacteria ....................................................................................................................... 15 1.1.1 Characteristics ........................................................................................................... 15 1.1.2 Mycobacterial pathogens: a re-emerging problem ............................................... 15 1.2 Molecular biology of mycobacteria: current knowledge and progress .................................17 1.2.1 Bacterial cloning hosts for expression of mycobacterial genes: gene expression in a heterologous background ..............................................................17 1.2.2 Mycobacteria as cloning hosts: gene expression in a homologous background .........................................................................................18 1.2.2.1 Mycobacteriophage and shuttle-phasmids ............................................ 19 1.2.2.2 Mycobacterial plasmids and shuttle vector technology ...................... 20 1.2.2.3 Insertion elements and transposons ...................................................... 21 1.2.3 Expression of foreign genes in mycobacteria .........................................................22 1.3 Gene regulation ..........................................................................................................................23 1.3.1 Initiation of transcription is catalysed by RNA polymerase: A principal step in the regulation of gene expression ..............................................23 1.3.2 Regulatory proteins ..................................................................................................24 1.3.3 Gene regulation in mycobacteria: Current knowledge and progress ................... 24 1.4 DNA damage in bacteria: a quick review ................................................................................26 1.5 DNA damage repair and tolerance systems in prokaryotes ....................................................27 1.5.1 DNA repair in prokaryotes ...................................................................................... 27 1.5.2 DNA damage tolerance systems ..............................................................................30 1.6 The RecA protein: a pivotal component of several cellular mechanisms .............................30 1.6.1 Homologous recombination ....................................................................................32 1.6.2 Recombinational or post-replication repair ............................................................35 1.6.3 Repair of double strand breaks ................................................................................37 1.6.4 The SOS response: Physiological consequences and regulation ..........................37 1.6.5 SOS Mutagenesis ...................................................................................................... 43 1.7 RecA mediated responses in mycobacteria .............................................................................43 1.7.1 A novel post-translational modification for the M. tuberculosis RecA protein ............................................................................................................ 44 1.7.2 Homologous recombination in mycobacteria .........................................................46 1.8 Aim s ........................................................................................................................................... 47 1.8.1 Background to the project ...................................................................................... 47 1.8.2 Specific aims of the project ....................................................................................48 2.0 M a t e r ia l s an d M e t h o d s ...................................................................................................... 49 2.1 Bacterial strains and plasmids; media and growth conditions ...............................................49 2.2 Production and purification of recombinant M. tuberculosis RecA protein ........................ 51 2.2.1 Over expression of M. tuberculosis recA by heat induction of E. co/z pEJ169 {TG2(pGPl-2)} ............................................................................ 51 2.2.2 Production of a crude preparation of recombinant M. tuberculosis RecA protein by differential precipitation .............................................................51 2.2.3 Purification of a crude preparation of recombinant M. tuberculosis RecA protein by SDS-polyacrylamide gel electrophoresis .................................51 2.3 Production of polyclonal