Conformational changes in DNA and MutS during mismatch repair in Escherichia coli, analyzed by fluorescence spectroscopy [Elektronische Ressource] / vorgelegt von Michele Cristóvão

Cristovão, Dominique Michele

Sujets

• Biologie

Informations

• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2009
• Nombre de lectures: 10
• Langue: English
• Poids de l'ouvrage: 7 Mo

Extrait

Conformational changes in DNA and MutS during
mismatch repair in Escherichia coli, analyzed by
fluorescence spectroscopy
Inauguraldissertation

zur Erlangung des Grades
Doktor der Naturwissenschaften
Dr. rer. nat.
Des Fachbereiches Biologie und Chemie
der Justus-Liebig Universität
Vorgelegt von

M. Sc. Michele Cristóvão

Gießen, 2009

The present study has been carried out at the Institute of Biochemistry, Justus-Liebig
University Giessen, between March 2006 and March 2009, under the supervision of Prof. Dr.
Peter Friedhoff and Prof. Dr. Alfred Pingoud.











Dean Prof. Dr. P. Schreiner
Institut für Organische Chemie
Justus-Liebig-Universität
Heinrich-Buff-Ring 58
35392 Giessen
Advisor Prof. Dr. Peter Friedhoff
Institut für Biochemie
Justus-Liebig-Universität
Heinrich-Buff-Ring 58
35392 Giessen
Co-advisor Dr. Mark Szczelkun
Department of Biochemistry
School of Medical Sciences
University Walk
Bristol BS8 1TD, UK


Acknowledgements

I would like to thank:
Prof. Dr. Alfred Pingoud, for giving me the opportunity to work in his lab, for the
constructive critics and suggestions.
Prof. Dr, Peter Friedhoff, my supervisor, for his guidance, amazing patience, motivation,
insightful discussions, interest and availability.
Dr. George Silva, G.---, thank you for all your help with my constant computer fights, for
your motivation to do good science, for your love for science, time for discussions, patience
and your friendship. Thanks for pushing me to do better.
Jasmina (Jazzy ) for all you help, discussions and laughs. It wouldn’t have been the
same without you, specially our time in Düsseldorf.
Dr. Silke Silva, Silke, for your friendship and patience and interesting nights playing
Guitar Hero .
Laura and Jada, pity you are not here anymore, but it was great having you around in the
beginning of my life in Giessen, with all the gym time and even some nights out.
Lena and Daniel, thanks for the fun times playing wii. That was always great!
To all my friends, who I left in different places, but that have an important role in my life.
To tall the “Marie Curie people”, specially Chris and Kara, for all the interesting
meetings.
And of course to all the MMR group, for the interesting working environment.

I specially would like to thank my parents, Anne e Manuel Cristóvão, for their spirit of
sacrifice, strength, example, love and support. Adelina, Amandio and Íris for always being
there for me. A very special thanks to Heinz Abels, whose enormous curiosity was always
very inspiring and motivating.

I would also like to thank the following collaboration partners:
Evangelos Sisamakis, Dr. Paul Rothwell and Prof. Claus Seidel, Institute of Molecular
Physical Chemistry, Univerisity of Düsseldorf. In particular, I would like to thank Evangelos
for his invaluable help and availability.
PD Dr. Ute Curth, Medizinische Hochschule, Hannover, Germany
I would like to thank the Marie Curie Research Training Network “DNA Enzymes” for
funding my work.

Erklärungen

Hiermit versichere ich, die vorliegende Arbeit selbständig verfasst und keine andere als
die angegebenen Hilfsmittel benutzt zu haben. Stellen, die ich anderen Arbeiten und
Veröffentlichugen dem Wortlaut oder Sinn entsprechend entnommen habe, sind durch
Quellenangaben gekenzeichnet.





Giessen, den 24. März 2009

Summary


Summary

Conformational changes both in DNA and MutS during the initial steps of DNA mismatch
repair system and during the ATPase cycle were analyzed using state-of-the-art fluorescence
techniques, down to the single molecule level. The work is presented in three chapter focusing on
different aspects of the mismatch repair initiation process.

MutS binds mismatches with preferred orientations
MutS scans the DNA for base-base mismatches and small insertion-deletion loops. A
hallmark of the mismatch recognition mechanism is DNA kinking by 45º-60º as observed in the
co-crystal structure of MutS-DNA complexes from bacteria to man. The change in the distance of
two positions in the DNA upon kinking was exploited by FRET (Fluorescence Resonance Energy
Transfer). 16 different 42bp oligonucleotides containing all possible base-base combinations (four
Watson-Crick and 12 mismatches) in a central position, with an acceptor fluorophore on the tope
strand and a donor on the bottom strand, were tested for DNA binding and kinking by MutS using
an in-solution FRET assay. In addition, selected base-base mismatches were analyzed for changes
in fluorescence anisotropy of the donor and acceptor fluorophores to probe the binding orientation
of MutS. Finally, single molecule Multiparameter Fluorescence Detection (smMFD) was used to
analyze the binding and bending modes of MutS at highest resolution. The results demonstrate
that MutS binds certain mismatches with a preferred orientation which can be analyzed by the
asymmetry introduced via the fluorescence dyes attached to the DNA. These observation were
corroborated by FRET analysis between fluorophore-labeled DNA and MutS labeled with
fluorophores in the clamp or the connector domain. The preferential binding orientation may have
important impact on the coupling of mismatch repair and replication, in particular on the
mechanism involving directed loading of the heterodimeric MutS α by interaction with the
replication factor PCNA.

Nucleotide influence of the DNA binding
MutS belongs to the ABC ATPase family (ATP-Binding Cassette) whose ATPase activity is
largely coupled to the dimerization of the ATPase domains. A crucial feature of these enzymes is
the coupling of ATP-binding and hydrolysis to a substrate binding site, i.e. DNA in case of MutS..
The double labeled G:T oligonucleotide was used to determine the binding and bending kinetics
of MutS to DNA in the presence of ADP, ATP and ADPnP. In addition, the influence of
nucleotide on the MutS-induced DNA bending was analyzed using smMFD to determine the
bent/kinked populations present with each nucleotide. The results showed that the association of
MutS to DNA in the presence of ADP involves at least a two step mechanism, possible a fast
5
Summary


binding/bending step followed by a kinking at the mismatch. A quantitative analysis of the FRET
population showed that the DNA with MutS almost homogeneous forming mainly a ADP
kinked/bent complex. In contrast, complex formed in the absence of nucleotide or in the presence
of ATP are more heterogenous involving at least two complexes, one of which is kinked/bent
whereas other are either unbent or bound not at the mismatch. Pre-steady state kinetic analysis of
MutS-DNA association in the presence of ATP and MutS-DNA dissociation in the presence of
ATP, ADP or ADPnP revealed distinct phases depending on the nucleotide state of the starting
complex. The data obtained by the smMFD and the fast kinetics of DNA binding and bending
was inserted into a model for DNA/ATP binding by MutS.

Communication between ATPase domain and clamp domain
Several conformational changes that MutS undergoes during the DNA binding and ATPase
cycle have been proposed, however, little experimental data is available proving these hypothesis.
In the present work conformational changes in MutS were monitored using a FRET analysis
employing single-cysteine variants of MutS. To simplify the data analysis a fully functional
R449C/D835Rsingle-cysteine dimer variant of MutS (MutS ) was generated thereby avoiding
complication due to the formation of tetramers in case of wild type MutS. The work presented
here shows that single-cysteine mutants of MutS could be fluorescently labeled with one or two
fluorophores suitable for FRET analysis without affecting the function of the protein, e.g. in DNA
binding, mismatch recognition and mismatch-provoked MutH activation.. Sedimentation velocity
analysis and in-solution FRET measurements demonstrated that the fluorescently labeled
R449C/D835RMutS forms stable dimers in the presence of DNA or nucleotide. Binding of the non-
hydrolyzable ATP-analogue ADPnP resulted in a closed, compact form of MutS which is unable
to bind to DNA. The dynamics of conformational changes in the clamp domain upon DNA and/or
nucleotide binding were monitored using stopped-flow. Based on these data the clamp domain of
MutS exists in at least four different states, i.e. flexible/open in the ADP-bound form, tight/closed
in the ATP-bound form, wide/closed in the mismatch/ADP-bound form and tight/close in the
DNA/ATP-bound form.
The data obtained from the fluorescence analysis using double labeled DNA, labeled
DNA/labeled MutS and double labeled MutS were included in model for the DNA binding and
ATPase cycle of MutS.
6
Zusammenfassung
In der vorliegende Arbeit wurden Konformationsänderungen in DNA und MutS, die in den
initialen Schritten des DNA-Fehlerreparatursystems und des ATPase Zyklus von MutS
vorkommen, mitt