Regulation of the ups pili system involved in DNA damage response in sulfolobus

Regulation of transcriptional initiation by general transcription factors .... 37 Effect of UV irradiation on Sulfolobus acidocaldarius and involvement of the general transcription fac

Regulation of the Ups pili system involved in

DNA damage response in Sulfolobus

Inaugural-Dissertation

zur Erlangung des Doktortorwürde der Naturwissenschaften (Dr rer nat.) am

Fachbereich Biologie der Albert-Ludwigs-Universität

Freiburg im Breisgau

Thuong Ngoc Le

geboren am 19.08.1988 in Thai Nguyen, Vietnam.

Regulation of the Ups pili system involved in

DNA damage response in Sulfolobus

Inaugural-Dissertation

zur Erlangung des Doktortorwürde der Naturwissenschaften (Dr rer nat.) am

Fachbereich Biologie der Albert-Ludwigs-Universität

Freiburg im Breisgau

Thuong Ngoc Le

geboren am 19.08.1988 in Thai Nguyen, Vietnam

The light microscopy picture on the cover shows S acidocaldarius cells forming aggregates due to

UV irradiation The picture was taken by Thuong Ngoc Le

Die vorliegende Arbeit wurde von Febuary 2014 bis August 2014 am Max-Planck Institut für terrestrische Mikrobiologie in Marburg und von September 2014 bis Marz 2018 an der Albert-Ludwigs-Universität Freiburg in der Arbeitsgruppe von Frau Prof Dr Sonja-Verena Albers durchgeführt

Dekanin der Fakultät für Biologie: Prof Dr Bettina Warscheid

Promotionsvorsitzender: Prof Dr Andreas Hiltbrunner

Betreuer der Arbeit:

Referent: Prof Dr Sonja-Verena Albers

Koreferent:

Drittprüfer:

Datum der mündlichen Prüfung:

The results that I have achieved during my Ph.D., which are described in this thesis, are published or to be published in the following peer review articles:

1 Thuong Ngoc Le, Alexander Wagner and Sonja-Verena Albers A conserved

hexanucleotide motif is important in UV-inducible promoters in Sulfolobus acidocaldarius

Table of contents

1 INTRODUCTION 1

1.1 Transcription in archaea: a mosaic of eukaryotic and bacterial features 2

1.1.1 Basal transcriptional machinery in archaea 2

1.1.2 Regulation of transcription in archaea 5

1.1.2.1 Regulation of transcriptional initiation by general transcription factors 5

1.1.2.2 Regulatory motifs in archaeal promoters 5

1.1.2.3 Gene-specific transcriptional regulators: repressors, activators 6

1.1.2.4 The role of chromatin binding proteins in transcription regulation 7

1.1.2.5 The regulatory role of non-coding RNAs (ncRNAs) in gene expression 8

1.2 The DNA damage response in hyper-thermophilic archaea 9

1.2.1 The DNA damage response (DDR) 9

1.2.1.1 UV - induced DNA damages 9

1.2.1.2 DNA repair mechanisms 10

1.2.2 The UV response in Sulfolobus is part of the DNA damage response 11

1.2.2.1 The hyper-thermophilic Sulfolobus 11

1.2.2.2 The Ups system in Sulfolobus 12

1.2.2.3 The Ced system 14

1.2.3 Regulation of the Ups and Ced system in Sulfolobus 14

1.2.3.1 Transcription factor B3 (TFB3) 14

1.2.3.2 Other players might be involved in regulation of the UV response in Sulfolobus 15

1.3 MoxR-like protein family 16

1.3.1 MoxR proteins’ characteristics and cellular functions 16

1.3.2 The moxR-vWA3 operon in Sulfolobus acidocaldarius 18

1.4 Scope of the thesis 19

2 RESULTS 20

Research article 1 21

A conserved hexanucleotide motif is important in UV-inducible promoters in Sulfolobus

acidocaldarius 21

Research article 2 37

Effect of UV irradiation on Sulfolobus acidocaldarius and involvement of the general transcription factor TFB3 in early UV response 37

Research article 3 72

Characterization of a MoxR AAA+ ATPase in Sulfolobus acidocaldarius 72

3 DISCUSSION 97

3.1 The role of the transcription factor TFB3 in the transcriptional regulatory network in response to UV-induced damage DNA in Sulfolobus 99

3.2 Transcriptional regulators and chromatin-binding proteins: their interplay and evolutionary relationship 103

4 Thesis summary 108

Zusammenfassung 109

References 110

Acknowledgment 116

Introduction

1 INTRODUCTION

Introduction

2

1.1 Transcription in archaea: a mosaic of eukaryotic and bacterial features

1.1.1 Basal transcriptional machinery in archaea

Forty years ago, based on the 16s rRNA gene sequences, archaea were recognized as the

third domain of life next to bacteria and eukaryotes (Woese et al., 1990; Woese & Fox,

1977) Since that milestone of evolution biology, a great number of studies have unveiled more unique characteristics of archaea that make them a separated domain (White, 2006; Cavicchioli, 2010; Werner & Grohmann, 2011; Albers & Meyer, 2011; Lindås &

Bernander, 2013; Karr et al., 2017) Nowadays, it is well established that archaea possess

a transcription apparatus resembling a simplified version of the eukaryotic RNA polymerase (RNAP) II system (Soppa 1999; Geiduschek & Ouhammouch 2005; Grohmann

& Werner 2011; Orell et al 2013; Karr 2014; Gindner et al 2014; Kessler et al 2015)

Studies on archaeal RNAP from Sulfolobus acidocaldarius delivered the first hint that archaea might initiate transcription in a eukaryotic manner (Zillig et al., 1979) Archaeal

RNAP is a protein complex composed of 13 subunits of which each individual subunit is

highly conserved and homologous to that of eukaryotic RNAP II (Langer et al., 1995; Grohmann et al., 2009) Subsequently, the canonical core promoter of archaea was

shown to harbor a TATA box, an AT-rich region located around -26 to -30 bp upstream of the transcription start site (TSS) Directly upstream of the TATA box is a purine-rich segment named transcription factor B recognition element (BRE) (Soppa 1999; Qureshi et

al 1997) There are other less defined DNA elements in archaeal promoters, such as the

initiator element (INR), and the promoter proximal element (PPE) (Peng et al., 2009a;

Soppa, 1999b) However, the presence of the INR and PPE varies among different groups

of archaea For example, the INR positioned within the initially transcribed region is hardly detectable in haloarchaeal promoters, but very pronounced in methanogens and Sulfolobales (Soppa, 1999b) The PPE located between the TATA box and the TSS has

been found primarily in Sulfolobus promoters (Peng et al., 2009a, 2011; Wurtzel et al., 2010)

Neither RNAPII nor RNAP can be recruited to promoters without the aid of transcription

factors (Blombach & Grohmann, 2017; Langer et al., 1995; Soppa, 1999a) So far, three

types of transcription factors involving initiation of archaeal transcription have been well studied They are TATA-binding proteins (TBPs), transcription factor B (TFBs), and

Introduction

transcription factor E (TFEs) Out of the three, TBP and TFB are necessary and sufficient

for promoter-specific transcription in vitro (Bell et al., 1998) Assembly of TBP and TFB on

the promoter recruits RNAP to the TSS and consequently forms the pre-initiation complex (PIC) TFE was initially shown to facilitate transcription initiation by enhancing TATA-box

recognition (Bell et al., 2001) Another study, however, demonstrated that TFE stabilizes the transcription bubble during elongation (Grünberg et al., 2007) These three archaeal

transcription factors are homologs of eukaryotic TBPs, TFBII, and TFEα, respectively (Bell

& Jackson 2000; Thomas & Chiang 2006; Werner & Weinzierl 2005; Grove 2013)

Archaeal TBPs are almost identical to the C-terminal domain of TBPs in eukaryotes (Soppa, 1999a, 2001) Additionally, their function is also equivalent to the role of eukaryotic TBPs Archaeal TBPs are about 180 amino acids long and consist of two direct repeats that are around 40% identical to each other (Soppa, 1999a) Transcription initiates by the recognition and binding of TBP to the TATA box Moreover, binding of TBP

helps to bend the promoter and recruits TFB to the BRE site (Bell et al., 1999a; Qureshi et

al., 1997; Soppa, 2001) Recently, a study revealed the differences of the lifetime of the

TBP– DNA interaction between the archaeal and eukaryotic system (Gietl et al., 2014) For instance, the eukaryotic DNA-TBP interaction follows a linear, two step-bending mechanism with an intermediate state having a distinct bending angle Here, TFBII helps

to stabilize the fully bent TBP– promoter DNA complex On the other hand, the bending of the archaeal promoter by TBP is a single step and TFB is strictly required for that process However, stabilization of the TBP–DNA complex by TFB does not seem to be a general mechanism, but probably an additional mechanism that mediates specificity among archaeal TATA-containing promoters (Gietl et al., 2014)

TFBs in archaea show significant similarity to eukaryotic TFIIB The protein consists of an terminal domain of 100-120 amino acids and a C-terminal domain containing two repeat sequences of around 90 amino acids (Soppa, 1999a) The C-terminal core domain and the helix-turn-helix (HTH) motif of TFB are responsible for interaction of TFB with TBP and binding

N-of TFB to the BRE, respectively (Bell & Jackson, 2000b; Qureshi et al., 1997; Soppa, 1999b) The crystal structure of TBP and the C-terminal core of TFB (TFBc) from P woesei in a complex

with a promoter containing TATA-box and BRE provided a detailed picture of the stereo specific interactions between the BRE and a helix–turn–helix motif in the C-terminal of TFBc

Introduction

4

(Littlefield et al., 1999) More importantly, the interaction of TFB with TBP-DNA serves as a

platform to recruit RNAP to the TSS (Nikos & Christos, 1999; Soppa, 1999a) The N-terminal domain of TFB harbors a zinc (Zn) ribbon motif that interacts with RNAP (Soppa, 1999a) In contrast to eukaryotes, opening of the archaeal promoter does not require energy (Hausner

& Thomm, 2001; Yan & Gralla, 1997) Archaeal TBP and TFB alone are capable of assisting RNAP in the formation of the transcription bubble (Hausner & Thomm, 2001)

Figure 1: A: Crystal structure of the archaeal ternary complex formed between TBP, TFBc (C-terminus of

TFB) and a TATA-box and BRE – containing oligonucleotide Molecules are coded in colors, pink and yellow:

DNA; green ribbon: TBP; magenta: TFBc (Littlefield et al., 1999); PDB Acc No 1D3U B: Formation of a

pre-initiation complex (PIC) in archaea Initially, TATA-binding protein (TBP) recognizes and binds to the TATA box, the transcription factor B (TFB) interacts with the TBP– DNA complex and the DNA–TBP–TFB complex

subsequently recruits RNAP and transcription factor E (TFE) C: Repressors regulate transcription by binding

to core promoter-overlapping sequences, thereby preventing access of the basal transcription factors TBP and TFB to promoters or of RNAP in a later stage of PIC assembly On the other hand, transcriptional activators function in the initial steps of PIC assembly by binding to the sequences upstream of the BRE

Figures B, C were adapted from (Peeters et al., 2015)

Archaeal TFE is a homolog of the α subunit of TFIIE TFE was shown to associate with

RNAP and stimulate DNA melting (Grünberg et al., 2007) The βTFE in archaea was

Introduction

recently identified and shown to interact with TFE (αTFE) but βTFE are not conserved in

all Archaea (Blombach et al., 2015)

1.1.2 Regulation of transcription in archaea

Regulation of transcription occurs at all steps from initiation to termination Nevertheless, regulation of transcriptional initiation is the most effective way to control gene expression Transcription regulation at initiation step involves promoter elements, general transcription

factors and gene-specific regulators (Balleza et al., 2009; Gehring et al., 2016; Karr, 2014)

1.1.2.1 Regulation of transcriptional initiation by general transcription factors

Regulation of transcription by general transcription factors (GTFs) is an important way to control transcription of genes In bacteria, numerous sigma (σ) factors help to modulate

gene expression in response to environmental stresses (Balleza et al., 2009) In

eukaryotes, the presence of four different RNAPs and multiple GTFs accomplish the global

transcription regulation (Lelli et al., 2012) Most archaeal genomes encode at least one

TBP and one TFB (Gehring, Walker, and Santangelo 2016) Some archaea have multiple

paralogs of GTFs For instance, Halobacterium NRC-1 1 has six TPBs and seven TFBs, which

theoretically could generate 42 different pairs of TFB-TBP to modulate the transcription

initiation (Ng et al., 2000) Indeed, it was shown that 7 out of 42 possible TFB–TBP pairs were used by Halobacterium NRC-1 and TBPe was the favorite TBP that interact with most TFBs (Facciotti et al., 2007) On the other hand, Methanosarcina acetivorans possesses three TBPs homologs (TBP1, TBP2, and TBP3) and one TFB (Galagan et al.,

2002) In this organism, TBP1 plays a greater role in gene expression than TBP2 and TBP3 However, TBP2 and TBP3 are important for optimal growth under growth-limiting acetate

concentrations (Reichlen et al., 2010)

1.1.2.2 Regulatory motifs in archaeal promoters

Regulators control the transcription of genes by binding to specific DNA sequences called binding motifs in promoters Generally, the location of the binding sites with respect to the core promoter (TATA box and BRE) determines the regulator as a repressor or

activator (Peeters et al., 2013) In most cases, archaeal repressors bind to sites which

overlap with or downstream of the BRE and/or TATA box Thereby, binding of repressors

Introduction

6

inhibits the PIC formation by preventing TBP, TFB or RNAP accessing to the promoters (Bell et al 1999; Dahlke & Thomm 2002; Lee et al 2008; Karr 2010; Keese et al 2010) In contrast, activators are often found to bind to sequences upstream of the core promoter

(Kessler et al., 2006; Ochs et al., 2012; Ouhammouch et al., 2003; Peng et al., 2009b)

However, the number of activators identified in archaea and the understanding of their mode of actions are still very limited in comparison to what has known about archaeal

repressors (Gehring et al., 2016; Karr, 2014; Peeters & Charlier, 2010)

Accompanied by the known transcription regulators, their binding sites on promoter regions were also identified as regulatory motifs The majority of motifs serve as binding sites of repressors, such as the TGM (Thermococcales-glycolytic-motif) in

Thermococcales, binding motifs of MDR1, NrpR or Phr (Bell et al., 1999b; Keese et al., 2010; Lee et al., 2005; Lie et al., 2010) Some upstream activating motifs were identified

as binding sites of activators such as ArnR, ss-Lrp, Ptr2, MalR (Lassak et al., 2013; Ouhammouch et al., 2003; Peeters et al., 2009; Wagner et al., 2014) Interestingly, the

ARA box, a DNA sequence in the promoter of gene encoding the arabinose-binding protein (AraS), was defined as an activating element, while the regulator is still missing

(Peng et al., 2009a, 2011) Nevertheless, most of the regulatory motifs share a common

feature of a semi-palindromic sequence with a number of less or non-conserved base

pairs in the center (Peeters et al., 2013) Exceptionally, the binding site of the heat shock regulator Phr in Pyrococcus furiosus is a non-palindrome (Keese et al., 2010)

1.1.2.3 Gene-specific transcriptional regulators: repressors, activators

Although possessing a eukaryotic-like basal transcription machinery, archaeal genomes contain an overabundance of putative bacterial like regulators rather than eukaryotic counterparts (Aravind & Koonin, 1999; Nikos & Christos, 1999) With a few exceptions, many archaeal regulators are similar to bacterial helix-turn-helix (HTH) containing-domain

proteins, others harbor the ribbon– helix–helix (RHH) motif, or the Zn ribbon (Aravind et

al., 2005; Aravind & Koonin, 1999) Transcriptional regulators with a HTH motif often bind

as dimers or higher oligomers to palindromic DNA sequences and contact to two consecutive

major groove segments aligned on the same face of the DNA helix (Aravind et al., 2005)

Introduction

In term of mechanism, archaeal repressors hamper transcription initiation by impairing promoter accessing of TBP and TFB through steric hindrance (Bell & Jackson, 2000a) Some repressors that bind to sites downstream of the TATA box inhibit the recruitment of RNAP to the TBP-TFB complex (Bell, 2005) Differences in repressing mechanisms between certain genes are probably due to their specific biological functions For

instance, MDR1 (metal-dependent transcriptional repressor 1) from Archaeoglobus

fulgidus inhibits RNAP recruitment to the promoter of an operon encoding MDR1, and

three components of an iron - importing ABC transporter system (Bell et al., 1999b) By

having pre-bound TBP and TFB on the promoter, RNAP could be rapidly recruited to

initiate transcription when needed This ability to respond rapidly might be crucial for A

fulgidus to maintain levels of vital metal ion co-factors (Bell & Jackson, 2000a)

Nevertheless, not all repressors in archaea follow these repressing mechanisms Some repressors can initially bind only upstream of the core promoter, then displace TBP and TFB by extending their bindings further downstream, as the case of RHH-containing AvtR

from Acidianus filamentous virus 6 (AFV6) and TrpY in Methanothermobacter

thermautotrophicus (Karr et al., 2008; Peixeiro et al., 2013)

Transcriptional activation in archaea is relatively less understood than repression However, it is certain that activation takes place at one of the initial steps of the PIC formation, either in helping binding of TBP, TFB, or RNAP by stimulating protein–protein

interactions (Kessler et al., 2006; Ochs et al., 2012; Ouhammouch et al., 2003; Paytubi &

White, 2009) In contrast to repressors that can function in combination with any promoter strength, activators are generally associated with weak promoters that have non-consensus BRE and/or TATA box or when the TFB possess a non-canonical Zn ribbon

(Ouhammouch et al., 2003; Paytubi & White, 2009; Peng et al., 2009a)

1.1.2.4 The role of chromatin binding proteins in transcription regulation

Archaeal genomes are compacted by either eukaryotic–like histones or bacterial-like

chromatin-associated proteins (Ammar et al., 2012; Reeve, 2003) In Crenarchaea, small

proteins like Alba, Cren7, Sul7, and CC1 compose the majority of chromatin binding

proteins (Peeters et al., 2015; Reeve, 2003) In contrast, homologues of the eukaryotic

histones H3 and H4 are the most abundant chromatin-binding proteins in Euryarchaea

Introduction

8

(Peeters et al., 2015; Reeve, 2003) Recently, a few studies have revealed the interplay of

archaeal chromatin binding proteins and basal TFs as well as gene-specific transcription

regulators in controlling gene expression (Ammar et al., 2012; Peeters et al., 2015; Xie &

Reeve, 2004) It was suggested that the binding competition of archaeal histones and TFs

could be a mechanism in regulation of gene transcription (Xie & Reeve, 2004) In M

thermautotrophicus, the HMtA2 histones inhibit transcription initiation by binding to the

site downstream of the TSS Since they facilitate the formation of a filament that extends

upstream then overlaps the core promoter (Xie & Reeve, 2004) In Methanococcus

jannaschii, the activator Ptr2 shares the binding site with histone tetramers in the

promoter of rb2 gene When Ptr2 is absence, binding of histones completely silence transcription of rb2; or repress the transcription at low level if TBP and TFB are still

capable to access the promoter Only when Ptr2 is present at high concentration, it

outcompetes with histones and initiates the transcription activation (Ouhammouch et al.,

2003) Vice versa, some transcriptional regulators not only function in gene regulation but also act as chromatin - like proteins at specific conditions such as the ss-Lrp and AbfR1 in

Sulfolobus (Li et al., 2017; Orell et al., 2013; Peeters et al., 2009) The biofilm regulator

Abfr1 in S acidocaldarius binds to DNA with high affinity but very low sequence specificity and induces strong DNA deformations (Li et al., 2017)

1.1.2.5 The regulatory role of non-coding RNAs (ncRNAs) in gene expression

Non-coding RNAs (ncRNAs) and their functions in gene regulation are well studied in both eukaryotes and bacteria Archaea species encode a plethora of ncRNAs molecules (Omer

et al., 2006, 2000; Wurtzel et al., 2010) In S solfataricus, the majority of identified

ncRNAs are cis – encoded antisense RNA transcripts which are fully complementary to

specific protein-coding genes like transposase genes (Tang et al., 2005) Thus, these

antisense RNAs may inhibit expression of the transposase mRNA to regulate transposition

of insertion elements (Tang et al., 2005) Another study in S solfataricus P2 demonstrated

a large portion of ncRNAs that are antisense to genes involving in ion transport Antisense

ncRNAs are speculated as the common regulatory mechanism in such genes in S

solfataricus (Tang et al., 2005; Wurtzel et al., 2010)

Introduction

1.2 The DNA damage response in hyper-thermophilic archaea

1.2.1 The DNA damage response (DDR)

To maintain genome integrity against DNA damage, bacteria and eukaryotes have evolved sophisticated pathways known as DNA damage response (DDR) (Kreuzer, 2013) DDR mechanisms rapidly detect DNA lesions, activate DNA repair proteins, and inhibit DNA replication and cell division till the DNA damage repair is complete (Khanna & Tibbetts, 2006) The SOS response is a well-known DDR in many bacteria It is a genetically controlled network that involves more than 40 independent SOS genes (Janion, 2008; Michel, 2005) The repressor LexA and inducer RecA play key roles in the regulation of the SOS response During normal growth, the LexA repressor binds to the SOS box in the promoter region of SOS genes and prevents their expression When DNA damage is present at high levels, LexA undergoes a self-cleavage reaction induced by the RecA filament (complex of RecA and ssDNA) then SOS genes can be expressed They are such as genes encoding for DNA repair enzymes, division inhibitor protein, and mutagenic DNA repair polymerase Pol V (Michel, 2005) The RecA filament also has a role in invasion of a homologous double-stranded DNA sequence and catalyze strand exchange (the key reaction of HR) (Janion,

2008; Kreuzer, 2013; Michel, 2005; Rastogi et al., 2010)

In eukaryotes, the cellular response to DNA damage is regulated and coordinated by the DDR signaling pathway, which is orchestrated by the ATM (ataxia-telangiectasia mutated),

and ATR (ATM- and Rad3-Related) (Finn et al., 2012; Marechal & Zou, 2013) Both ATM

and ATR are activated by DNA damage and DNA replication stress, but their DNA-damage specificities are distinct and their functions are not redundant (Marechal & Zou, 2013)

1.2.1.1 UV - induced DNA damages

Among DNA damaging sources, UV radiation is accountable for direct DNA lesions such as cyclobutane pyrimidine dimers (CPDs), hydrated pyrimidines, pyrimidine 6-4 pyrimidine

(6-4 PPs), and indirect damages like double-stranded DNA breaks (DSBs) (Rastogi et al.,

2010) The initial photoproducts such as CPDs, 6-4 PPs and reactive oxygen species (ROS) associated with transcription or replication blockage called “collapse of replication forks”

leads to production of DSBs (Limoli et al., 2002) UV radiation does not seem to produce

Introduction

10

DSBs directly but pyrimidine dimers and other photoproducts that lead to replication arrest and DSBs

Among all DNA lesions, DSBs are the most lethal since they affect both strands of DNA

and can lead to the loss of genetic material and cell death (Van Gent et al., 2001)

1.2.1.2 DNA repair mechanisms

Direct DNA lesions like CPDs or 6-4 PPs can be repaired by photo-reactivation involving photolyase that can specifically bind to the lesions and directly monomerize cyclobutane ring using light energy (Essen & Klar, 2006)

Unlike photo-reactivation, excision repair is a multiple-step pathway, where modified/damaged bases are removed by base excision repair (BER) or nucleotide excision repair (NER) (Sinha & Häder, 2002) In the BER pathway, damaged bases are removed by DNA glycosylases, and then deoxyribose is excised by a phosphodiesterase Following that repair, RNAP and DNA ligase fill and seal the gap on the DNA strand,

respectively (Wood, 1996; Sinha and Häder, 2002; Rastogi et al., 2010) The NER pathway

involves many steps and protein complexes that detect DNA damages, separate strands and assembly the repair machinery to incise DNA around the lesion The last steps

remove the damage-containing oligonucleotide and fill the gap (de Laat et al., 1999) NER

and BER are able to repair lesions located in one strand of dsDNA and remove them in a

“cut-and-patch”-mechanism In both ways, the undamaged complementary strand serves

as a template for repairing the damaged strand Non-homologous end joining (NHEJ) and homologous recombination (HR) are two independent pathways that can repair DSBs (Sonoda et al., 2006) NHEJ repairs DSBs by direct ligation of the broken ends The pathway relies on the specific DNA repair protein Ku70 and Ku80, DNA ligase IV and other

associated factors (Chiruvella et al., 2013) Repair of DSBs by NHEJ easily leads to errors

such as small insertions, deletions, substitutions at the broken site, and translocations when DSBs from different parts of the genome are joined (Heidenreich et al., 2003; Lieber, 2010) Repair of DSBs by HR employs an extensive sequence homology between the broken and template strands HR pathway initiates by the resection at the double strand break to remove the 5’ strand and produce the 3’ overhang The 3’ tail is responsible for strand

Introduction

invasion into a homologous dsDNA and serves as a primer for DNA synthesis leading to the formation of a joint molecule of four-stranded DNA termed Holliday junction (HJ) HJs are mobile and can move by the process that is called branch migration The HJs are removed by a helicase releasing nicked daughter duplex DNA that later is ligated (Grogan,

2015; Rastogi et al., 2010; White, 2011) In comparison to NHEJ, HR is considered as an

error-free DNA repair pathway (Sonoda et al., 2006)

Archaeal HR proteins are homologs to eukaryotic Rad50 and Mre11 and often encoded in

one operon together with protein HerA and NurA (Constantinesco et al., 2004) In

Pyrococcus furiosus, proteins Rad50, Mre11, HerA and NurA co-operatively catalyze the

DNA 3’ - end resection step (Hopkins & Paull, 2008) The strand invasion involves protein RadA, RadA paralogs and SSB forming the HJs Hjm helicase and Hjc nuclease probably

perform branch migration and resolve the HJ, respectively (Kvaratskhelia et al., 2001)

In a sense of genetics of archaeal DNA repair, it is likely that BER and HR are vital pathways in archaeal DNA repair while homologs of eukaryotic NER do not appear to play

a role in such process (She et al., 2017) Interestingly, in H volcanii Rad50-Mre11

prevents repair of DSBs by HR, perhaps to limit unrestrained chromosome rearrangements Instead, micro-homology-mediated end-joining is the primary pathway

to repair DSBs in H volcanii (Delmas et al., 2009)

1.2.2 The UV response in Sulfolobus is part of the DNA damage response

1.2.2.1 The hyper-thermophilic Sulfolobus

Crenarchaeal Sulfolobus species are found in extreme habitats where high temperature and UV irradiation constantly threat genome integrity (Brock et al., 1972) However, the mutation rate of Sulfolobus species is not higher than that of mesophilic organisms such

as Escherichia coli (Grogan & Hansen, 2003) In order to do that, Sulfolobus must have

evolved a very effective way to detect and repair DNA damages

Archaea lack LexA homologs, therefore, also a classical SOS regulon (Eisen & Hanawalt, 1999) In addition, in some hyperthermophilic archaea, RadA, a homolog of RecA protein,

is constitutively expressed and only moderately induced by DNA damage (Fröls et al., 2007; Götz et al., 2007; Lundgren & Bernander, 2007) Studies of DNA damage response

Introduction

12

done in Halobacterium NRC1 and Sulfolobus species showed that though their genomes

contain genes coding for bacterial and/or eukaryotic NER pathways, none of these set

genes exhibits significant up-regulation in response to DNA damage (Baliga et al., 2004; Fröls et al., 2007; Götz et al., 2007) Although Sulfolobus showed no increase in

transcription of DNA repair genes following UV irradiation, a repression of DNA

replication and chromatin proteins was still observed (Fröls et al., 2007; Götz et al., 2007)

More interesting was the significant up-regulation of gene clusters that are responsible

for biogenesis of UV-inducible pili of Sulfolobus (ups) and Ced DNA transfer proteins Both systems have been identified to play significant roles in the survival of Sulfolobus upon severe DNA damage (Fröls et al., 2007; Götz et al., 2007; van Wolferen et al., 2016)

Table1: Genes highly regulated by UV extracted from (Fröls et al., 2007; Götz et al., 2007)

SSO no saci no Annotations SSO no saci no Annotations

SSO691 saci_0568 CedA - DNA import SSO0911 saci_1374 CdvA ESCRT-III

SSO3146 saci_0568 CedA - DNA import SSO0910 saci_1373 CdvB ESCRT-III

SSO0152 saci_0748 CedB-DNA import SSO0909 saci_1372 CdvC ESCRT-III

SSO283 saci_0667 HerA like ATPase SSO0257 saci_0722 cdc6-1 cell division control

SSO0121 saci_1493 UpsX SSO5826 saci_0843 transcriptional regulator

SSO0120 saci_1494 UpsE-Type IV pili ATPase SSO2241 saci_0046 ATPase

SSO0119 saci_1495 UpsF-pilus assembly SSO0034 saci_0204 parA

SSO0117 saci_1496 UpsA-prepilin SSO2288 saci_0144 permease

SSO0118 saci_1496b UpsB-prepilin SSO3242 saci_1012 transcriptional regulator

SSO280 saci_0665 TFB3-transcription factor B SSO2750 saci_1228 conserved protein

SSO2395 saci_0951 hypothetical protein SSO2751 saci_1229 conserved protein

SSO0771 saci_0903 cdc6-2 cell division control SSO0881 saci_1416 ESCRT-III

1.2.2.2 The Ups system in Sulfolobus

The ups cluster is highly conserved in all sequenced Sulfulobales genomes (van Wolferen

et al., 2013) There are five proteins encoded in the ups cluster Except for UpsX, a protein

of unknown function, the remaining proteins are essential for the assembly of functional ups pili Like other type IV pili, the ups pilus consists of an ATPase (UpsE) hydrolyzing ATP

to power the pilus assembly UpsF, a membrane protein, anchors the pilus to the cell membrane Two pre-pilin subunits form the pilus filament: UpsA and UpsB The pre-pilins

Introduction

contain a type III signal peptide which is cleaved by pre-pilin peptidase PibD before the

pilins are assembled to the growing filament (Ajon et al., 2011; Fröls et al., 2008; van Wolferen et al., 2013) Under UV stress condition, Sulfolobus express ups pili enabling

cells to form cellular aggregates and subsequent to exchange DNA between cells in a

species-specific manner (Ajon et al., 2011; Fröls et al., 2007) Although UpsX does not play

a role in pilus formation, DNA exchange after UV irradiation was indeed reduced when

UpsX was absent (van Wolferen et al., 2013) DNA exchange between aggregated cells is

presumed for repair the DSBs through HR, which is supported by the fact that inducing reagents such as bleomycin and mitomycin-C also induce cellular aggregation

DSBs-(Fröls et al., 2007)

Figure 2: Upper: UV-inducible pili on electron micrographs of S solfataricus (A), S tokodaii (B) and S

acidocaldarius (C); Lower: Schematic overview of the ups gene cluster in different Sulfolobales The cluster

encodes UpsX, a protein with unknown function; UpsE, a secretion ATPase; UpsF, an integral membrane protein; and UpsA and B, two pilin subunits Homology found using SyntTax (Oberto 2013) is indicated by

similar colors Figures were adapted from (Ajon et al., 2011; van Wolferen et al., 2013)

Introduction

14

1.2.2.3 The Ced system

By using the ups pili, Sulfolobus can species-specifically form the connection between cells to then exchange DNA (Ajon et al., 2011; Fröls et al., 2008) However, archaeal DNA

transport systems have remained poorly understood Recently, the first report about the DNA importing system in Crenarchaea uncovered a DNA importing system named Ced -

Crenarchaeal system for exchange of DNA (van Wolferen et al., 2016) The Ced system

contains two membrane proteins CedA and CebB which were shown to be highly

upregulated upon UV (Fröls et al., 2007; Götz et al., 2007) CedA is a transmembrane

protein, predicted to form a pore in the membrane to transfer DNA, which is presumed to function analogous to protein VirB6 of bacterial type IV secretion (Chandran Darbari & Waksman, 2015) Interestingly, CedB is a homolog of VirB4 proteins that are AAA ATPases

of type-IV secretion systems in bacteria (Fullner, Stephens and Nester, 1994; Watarai, Makino and Shirahata, 2002) CedA and CedB are both essential for DNA exchange after

UV stress in S acidocaldarius Furthermore, this research also showed that the Ups

system functions separately from the Ced system, even though both systems are essential for DNA transport after UV irradiation The Ups system functions in formation of the species-specific connection between cells, whereas the Ced system subsequently

functions actively in DNA import (van Wolferen et al., 2016) The presence of the Ups and Ced systems in Sulfolobus suggests a novel DNA repair pathway involving UV-inducible pilus formation and intercellular DNA transfer(Ajon et al., 2011; van Wolferen et al.,

2016) Moreover, the high expression of these two systems after UV stress is part of the

Soppa, 1999; Bell et al., 2000; Soppa, 2001; Werner and Weinzierl, 2005) Sulfolobus

Introduction

genomes possess three paralogues of the tfb gene Two of them (tfb1 and tfb2) encode

full-length TFB proteins while TFB3 is a truncated protein that lacks the HTH domain, the

B finger and a large part of the core domain (Götz et al., 2007)

Microarray studies of UV response of Sulfolobus species demonstrated a programmed transcriptional response to DNA damage (Fröls et al., 2008; Götz et al., 2007) Interestingly, tfb3 was significantly up-regulated following UV treatment whereas no such changes were observed with either other basal transcription factors or RNAP (Fröls et al., 2007; Götz et al., 2007) TFB3 activates transcription of several genes in vitro in the

presence of TFB1 and TBP (Paytubi & White, 2009) It might be due to the fact that TFB1

from Sulfolobus has the non-canonical Zn ribbon motifs that could result in a relatively weak interaction with RNAP (Dixit et al., 2004; Paytubi & White, 2009) TFB3, on the other

hand, has a conserved Zn ribbon domain, which could supplant that of TFB1 to provide full activation of transcription To do that, TFB3 needs the TBP-TFB1 complex properly assembled on promoters Hence, TFB3 possibly served as a molecular bridge between RNAP and the ternary complex, bringing them in close proximity to allow the activation of transcription (Grohmann & Werner, 2011; Paytubi & White, 2009) Taken together the great induction of TFB3 after UV stress and its ability to activate transcription suggest TFB3 probably functions as a specialized regulator that stimulates transcription in response to environmental stresses, such as UV irradiation Possibly, the Ups and Ced

systems in Sulfolobus could be under regulation of TFB3

1.2.3.2 Other players might be involved in regulation of the UV response in Sulfolobus

Besides TFB3, the regulator Sa-Lrp was also reported to be involved in the reaction of S

acidocaldarius to UV stress This regulator belongs to the Lrs14 family whose members

play important roles in cell motility and biofilm formation (Li et al., 2017; Orell et al., 2013) In the sa-Lrp deletion strain, S acidocaldarius cells are still able to form UV-

induced aggregations but the size of aggregates is significantly impaired Further, Sa-Lrp

was shown to bind to the promoters of the ups genes However, when Sa-Lrp is absent, except upsA no significant changes in transcription of ups genes was observed (Vassart et

al., 2013) It is likely that Sa-Lrp only plays a minor role or collaborates with still-missing

major players to drive transcription of the ups cluster

Introduction

16

The ups pili, the archaella, and the aap pili in Sulfolobus resemble type IV pili of bacteria (Lassak et al., 2012; Makarova et al., 2016; Ng et al., 2008) So far, only the regulation of the archaellum has been being extensively studied (Ding et al., 2016; Lassak et al., 2013;

Li et al., 2017; Reimann et al., 2012) The archaellum operon is under the control of

proteins such as Lrs14, vWA domain-containing proteins (ArnB), and other regulators

that form the archaella regulatory network (Arn) (Chaudhury et al., 2016; Reimann et al.,

2012) The vWA domain-containing proteins are well known to associate with proteins of the MoxR-ATPase family (Snider & Houry, 2006) In all Crenarchaea and some Euryarchaea that exhibit the T4P system, the pair of gene encoding MoxR–vWA domain carrying proteins is always co-occurrence It would be an alternative approach to study the vWA containing proteins and the associated MoxR in a relationship with the T4P pili such as the ups pili

1.3 MoxR-like protein family

1.3.1 MoxR proteins’ characteristics and cellular functions

MoxR proteins belong to one clade of the AAA+ ATPase (ATPases Associated with various cellular Activities) family Members of the AAA+ family are involved in protein/DNA remodeling or degradation where the energy of ATP hydrolysis is coupled to drive

mechanical works (Miller & Enemark, 2016; Neuwald et al., 1999; Olivares et al., 2015;

Sauer & Baker, 2011) Unlike others clades of AAA+ family that are found in all organisms, MoxR subfamily so far only has been identified in bacteria and archaea (Iyer et al., 2004) MoxR proteins are usually co-occurred with proteins that carry the metal-binding von Willebrand factor A (vWA) domain (Snider & Houry, 2006; Wong & Houry, 2012) Very often, they are encoded in one operon, which indicates the functional link between these two proteins Phylogenetically, MoxR proteins are divided into seven subfamilies Proteins belonging to the MRP- and RavA-subfamilies are well studied among all the identified MoxR (Snider & Houry, 2006)

MoxR proteins have an AAA+ domain composed of the α-β-α subdomain and the all-α subdomain (Snider & Houry, 2006) The α-β-α core domain harbors the canonical Walker

A and Walker B motifs, sensor 1 and the Arg finger (Snider et al., 2008) The all-α subdomain contains the sensor 2 motif and a conserved Arg interacting with the γ-

Introduction

phosphate of the bound ATP (Snider et al., 2008) Proteins of the MoxR family have the helical inserted before the sensor 2 The presence of the inserted helical places the all- α subdomain positioned at the back side of the α-β-α core, not forms a ‘’lid’’ over the top α-β-α core like others AAA+ proteins (Miller & Enemark, 2016)

α-In bacteria, MoxR proteins are shown to act as molecular chaperones in the maturation of

protein complex such as the methanol dehydrogenase (MDH) in in Paracoccus

denitrificans (Van Spanning et al., 1991) Other MoxRs are involved in cell shape

maintenance, stress response and pathogenicity (Bhuwan et al., 2016; Dieppedale et al., 2011; Wong & Houry, 2012) In Rhizobium leguminosarum, MoxR plays an important role

in maintaining cell envelope integrity Disruption of moxR in this plant pathogen leads to

aberrant cell size and increased sensitivity of membrane to some disruptive agents

(Dieppedale et al., 2011) A MoxR protein identified in Francisella tularensis is important

for tolerance to oxidative stress, acid stress, heat stress The cluster genes of which

FTL_0200 is encoded also is likely inducible by the heat shock associated transcriptional

regulator r32 The insertion mutant of FTL_0200 significantly impairs infection ability of F

tularensis (Dieppedale et al., 2011) Protein RavA from E coli K-12 has been extensively

characterized (Snider et al., 2006; Wong et al., 2017) Its corresponding vWA

domain-containing protein is known as ViaA RavA forms hexamer and exhibits ATPase activity that is enhanced by the presence of ViaA RavA was shown to associate and modulate the

inducible Lysine decarboxylase (Ldcl) that is responsible for acid stress response in E.coli (El Bakkouri et al., 2010; Kandiah et al., 2016) Moreover, interaction of RavA with Ldcl

prevent the binding of ppGpp, an alarmone which inhibit the activity of Ldcl (El Bakkouri

et al., 2010) Recently, the activity of fumarate reductase complex has been found under

control of RavA and ViaA interaction (Wong et al., 2017)

In archaea, two MoxR proteins have been characterized so far, despite the widespread of this family in all archaeal lineages The protein p618, a RavA family member, from the

crenarchaeal Acidianus two-tailed virus (ATV), was found to interact with the vWA

containing-domain protein p892 and suggested to play a crucial role in the dynamic of

extracellular tail development of the ATV virion (Scheele et al., 2011) The in vitro study of other MoxR in euryarchaeon Thermococcus kodakarensis revealed that KOD1 (TkMoxR)

forms hexameric and dodecamer conformations TkMoxR is able to bind to dsDNA

Introduction

18

molecules of various lengths in the presence of ATP However, only the dodecameric MoxR could catalyze dsDNA decomposition to form and release ssDNA The study indicates a helicase activity of TkMoxR which might involve in gene expression control

(Pham et al., 2014) Overall, the functions of proteins MoxR in archaea relatively remain

unidentified

1.3.2 The moxR-vWA3 operon in Sulfolobus acidocaldarius

S acidocaldarius genome contains one moxR gene (saci_0976) directly adjacent to a gene

encoding for a vWA domain – containing protein (saci_0977) in a predicted operon (Cohen et al., 2016) According to a previous study, Saci_MoxR belongs to the RavA subfamily where a protein such as RavA was extensively studied in E.coli (Wagner, Ph.D

dissertation 2017, Snider and Houry 2006; Wong and Houry 2012) The vWA protein

encoded in the moxR operon is one of three vWA domain-containing proteins in S

acidocaldarius The other two are ArnB, the repressor of the archaellum operon and

vWA2 encoded in the same cluster with ArnB but has no effects on the archaellum

regulation (Reimann et al., 2012) Therefore, the vWA domain-containing protein associated with MoxR now is named vWA3 A synteny analysis reveals that moxR

–vWA operon is highly conserved in Sulfobales and Crenarchaea Around the

cluster are genes encoding the single strand binding protein (SSB) and

components of a succinate dehydrogenase (SdhABCD) (Cohen et al., 2016)

Scope of the thesis

1.4 Scope of the thesis

UV radiation is one of the most dangerous threats to genome integrity of all living organisms especially those who inhabit extreme harsh habitats like hyper-thermophilic

archaea Microarray studies in Sulfolobus species following UV irradiation revealed a

transcriptional response to DNA damages Remarkable was the high upregulation of genes encoding proteins of the Ups and Ced system that are essential for DNA transport after UV irradiation Findings of the Ups and the Ced system suggest a novel DNA repair pathway involving UV-inducible pilus and intercellular DNA transfer However, the mechanism(s) that modulates transcription of genes encoding for the Ups, Ced systems, and other UV-regulated genes still has been under-investigated Interestingly, most archaeal transcriptional regulators are similar to bacterial counterparts while the components of transcription machinery in archaea are homologous to the RNAP II in eukaryotes In other words, transcription in archaea is a mosaic of eukaryotic-like apparatus and a bacteria-like paradigm

During the time of my Ph.D, I have attempted to find out and understand the regulatory mechanism that modulates the transcription of genes regulated by UV irradiation The scope of my thesis is to answer some of the important questions:

+ What promoter element(s) and regulators are important to transcription of the ups cluster? How are transcriptions of the ups and ced genes regulated in response to UV

stress?

+ Within this thesis, I also extended the work in investigating the function of protein

MoxR in Sulfolobus that was identified as the interaction partner of UpsX

Results

20

2 RESULTS

Research article 1 Results

Following UV irradiation, Sulfolobus species employed a novel pathway to repair damaged DNA,

which involves the Ups pili and the Ced system In this article, we have revealed the DNA motif

that plays important role in promoters of ups genes and other genes whose transcriptions were

altered after UV expose We had employed a β-galactosidase reporter gene assay to screening

varied promoter’s truncations of ups genes, which helped us define the important region in these

promoters In addition, we generated point mutations on the target region to study the consensus nucleotides that contributed to UV-induced transcription of genes harboring these promoters Eventually, we identified a hexanucleotide motif presented in promoters of most UV-up-regulated genes and genes showed down-regulation after UV treatment Mutation of the motif in

promoters of upsX and upsE gene resulted in phenotypes that are similar to effects caused by

deletion mutants of respective genes

Statement of the own participation

Thuong Ngoc Le (TNL) and Alexander Wagner (AW) were performed experiments under the supervision of Sonja-Verena Albers (SVA) TNL created plasmids, mutant strains and performed reporter gene assay, qRT-PCR and microscopy The bioinformatics analysis and DNA transfer assays were performed by AW and TNL The data analysis and manuscript preparation were done

by TNL, AW, and SVA

Research article 1 Results

22

Research article 1 Results

Research article 1 Results

24

Research article 1 Results

Research article 1 Results

26

Research article 1 Results

Research article 1 Results

28

Research article 1 Results

Research article 1 Results

30

Research article 1 Results

Research article 1 Results

32