Role of DisA and c-di-AMP in the DNA damage response of exponentially growing Bacillus subtilis ...

154 Pages·2015·16.46 MB·English

Universidad Autónoma de Madrid (UAM)
Faculty of Science
Department of Molecular Biology

Role of DisA and c-di-AMP in the DNA damage response of
exponentially growing Bacillus subtilis cells

PhD Thesis
Carolina Gándara
Madrid 2015

Thesis presented by Carolina Gándara to obtain the degree of Doctor of
Philosophy in Science at the Universidad Autónoma de Madrid (UAM),
Madrid, Spain.

This work has been performed at the Spanish National Biotechnology
Centre (CNB-CSIC) under the supervision of Dr. Juan Carlos Alonso.

Aos meus queridos pais

Abstract
Cells are constantly subjected to DNA lesions, which can result in replication fork stalling or
collapse. A variety of DNA repair pathways have evolved to respond to the different types of
exogenous and endogenous DNA damaging agents. A complex regulation exists to coordinate
these processes, which are called DNA damage response (DDRs). DisA was initially described
as a DNA damage checkpoint protein that ensures the genome integrity of Bacillus subtilis
spores. However, basal levels of DisA were detected in vegetative cell extracts and highly
dynamic foci were observed in vegetative cells expressing DisA-YFP under its native promoter.
Since DisA is also present in non-spore forming bacteria, a more general role for DisA, that is
not restricted to sporulation, was proposed.
In this work, it was demonstrated that c-di-AMP and the diadenilate cyclases (DAC), DisA
and CdaA, contribute to elicit repair of stalled and collapsed forks, in exponentially growing B.
subtilis cells. Absence of DisA or decreased intracellular levels of c-di-AMP impaired survival
to alkylation DNA damage induced by methyl methanesulfonate (MMS). On the other hand,
CdaA is involved in oxidative damage repair. The radA and disA functions were epistatic for
MMS and showed also epistasis with functions involved in Holliday junction (HJ) processing.
Both radA and disA were epistatic with recG and recU, while non-epistatic with ruvAB. In vivo,
DisA-YFP foci lost its dynamics in the presence of unresolved HJs (ΔrecU context).
Purified DisA showed DAC activity with preference for ATP over dATP to produce c-di-
AMP. DisA could bind different DNA structures without specificity, although only highly
stacked HJs could modulate DisA c-di-AMP synthesis. For the first time it was demonstrated
that RadA/Sms could bind DNA, in the absence or presence of ATP, and with more affinity for
single-stranded (ss) DNA and HJs. In vitro assays showed that DisA strongly inhibits RecG and
RuvAB helicase activities. DisA also negatively affects RecU resolvase activity and RecA-
mediated strand exchange reactions. Altogether, these results indicate that, in stalled forks
induced by the presence of alkyl adducts, DisA modulates the DDR by binding branched DNA
and reducing the synthesis of c-di-AMP. More precisely, DisA modulates recombinational
DNA repair by protecting branched recombination intermediates, and therefore preventing fork
reversal, HJ resolution and strand exchange. It is likely that DisA stimulate other specific DNA
repair pathways, such as BER, and favours TLS since slightly lower mutation frequencies were
detected in the absence of DisA.

Abbreviations

AS Ammonium sulphate
K Apparent dissociation constant
Dapp
ATP Adenosine triphosphate
LB Luria-Bertani medium
BER Base excision repair
LC-MS/MS Liquid chromatography-tandem
BSA Bovine serum albumin mass spectrometry
c-di-AMP Cyclic diadenosine LC Lethal concentration to kill 99%
99
monophosphate
MALDI-TOF Matrix-assisted laser
CFU Colony-forming unit desorption/ionization time-of-
CNB Centro Nacional de flight mass spectrometer
Biotecnología min Minutes
DAC Diadenylate cyclase MMC Mitomycin C
DAPI 4’, 6-diamidin-2-phenylindole MMR Mismatch repair
dATP deoxyadenosine triphosphate MMS Methyl methanesulfonate
DDR DNA damage response Nal Nalidixic acid
DDT DNA damage tolerance NER Nucleotide excision repair
DMSO Dimethyl sulfoxide NHEJ Non-homologous end joining
DNA Deoxyribonucleic acid Ni-NTA Nickel-nitrilotriacetic acid
DNaseI Deoxyribonuclease I nt Nucleotides
DSB Double-strand break OD Optical density
dsDNA Double-stranded DNA PAGE Polyacrylamide gel
DSS Disuccinimidyl suberate, electrophoresis
Suberic acid PDE Phosphodiesterase
DTT Dithiothreitol PEI Polyethyleneimine
EDTA Ethylenediaminetetraacetic acid RNA Ribonucleic acid
EMSA Electrophoretic mobility shift Rpm Revolutions per minute
assay
SDS Sodium dodecyl sulphate
H O Hydrogen peroxide
2 2 ssDNA Single-stranded DNA
HJ Holliday Junction
SSG Single-strand gap
HhH Helix-hairpin-Helix
TAE Tris-acetate EDTA
HR Homologous recombination
TLS Translesion synthesis
IPTG Isopropil-β-D-1-
wt Wild-type
tiogalactopiranósido
YFP Yellow fluorescent protein
kDa Kilo-dalton