Download University of Groningen Assembly dynamics of supramolecular

University of Groningen
Assembly dynamics of supramolecular protein-DNA complexes studied by singlemolecule fluorescence microscopy
Stratmann, Sarah
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to
cite from it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date:
2017
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Stratmann, S. (2017). Assembly dynamics of supramolecular protein-DNA complexes studied by singlemolecule fluorescence microscopy [Groningen]: Rijksuniversiteit Groningen
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the
author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately
and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the
number of authors shown on this cover page is limited to 10 maximum.
Download date: 03-08-2017
Chapter 1
Introduction
Sarah Stratmann
9
Chapter 1
1.1 DNA metabolism
“14% N, 5.8% P, 1.8% S.” This result of the chemical analysis of purified nuclei from leucocytes by Friedrich Miescher in 1871 (1) heralded a new era of research in physiological
chemistry. It let the author speculate about a so far unknown chemical moiety that he named
‘nuclein’, richer in phosphor and nitrogen than any other known protein. As it turned out,
Miescher succeeded for the first time to extract DNA from cell nuclei. Following Miescher’s
work, Albrecht Kossel decoded the chemical groups of nucleic acid, the five bases adenine,
thymine, guanine, cytosine, and uracil. Remarkably, Kossel realized that proteinaceous mass
was co-purified with the nucleic acids from nuclei, indicating a more complex and perhaps
biologically relevant structure. He suggested a novel class of nucleic-acid associating proteins
that he named histones (2). He postulated that these basic proteins, rich in lysine and arginine, might have transformed from ordinary proteins to interact with nucleic acids in ‘chromioles’ (later called chromosomes) through ionic interactions (3).
Today we know that DNA-interacting proteins play roles in every aspect of DNA metabolism:
A machinery of enzymes replicates DNA, specialized enzymes constantly repair errors in
the bases to prevent genomic mutations, structural proteins package DNA into three-dimensional structures, and nucleases degrade DNA in the process of cell death. Besides all these
enzymes involved in processing DNA, a large number of proteins and enzymes are involved
in transcription and translation and the vast regulatory network associated with it.
This thesis deals with two topics in DNA metabolism, investigated using biochemical as well
as single-molecule approaches: Assembly of DNA-replication complexes in E. coli, and the
sensing of pathogenic DNA in mammalian systems. These two processes represent illustrative
examples of the maintenance and processing of genomic material and the regulation required
to control these processes. DNA replication is a highly regulated process in all organisms.
In prokaryotes, nutrient supply represents a major factor triggering replication, whereas in
eukaryotes, cell-cycle factors determine whether the cell-division phase is to be entered. The
identification of pathogenic DNA also presents a tightly regulated mechanism in the context
of propagation of genomic material, albeit a negative one. Eukaryotic cells have developed
specific sensors that detect DNA and other forms of nucleic acid belonging to invading pathogens and that prevent their replication in the context of an inflammatory response. In the
following sections I will provide a brief background summary on both topics and define the
various research questions as a context for the work I present in this thesis.
1.2 DNA replication
Organisms have developed complex strategies to regulate the timing of replication initiation,
to ensure that the replication of genomic material is timed correctly in the context of the
cell-division cycle. In eukaryotes, multiple enzymes are involved in initiating the formation
of a large number of replication complexes on various positions on the chromosomal DNA.
The key proteins in this process are subject to a variety of cell-cycle dependent activation
processes, such as phosphorylation by cyclin-dependent kinases leading to protein ubiquiti10
DNA replication
B
A
oriC
oriC
Bidirectional fork
progression
DnaA boxes
AT domains
oriC
DnaA
HU
IHF
DnaC
Ter sites
Ter sites
DnaB
Pre-priming complex
Figure 1: DNA replication in E. coli. A) At the oriC sequence, two replication complexes are established that progress
bidirectionally along the chromosome, until they are stopped at the termination (Ter) sites. B) The initiator protein
DnaA oligomerizes at the oriC, unwinding the AT-rich domains. Two complexes that each contain a DnaB helicase
and DnaC helicase loader assemble onto the single-stranded region and trigger the formation of a pair of replisomes.
nation and degradation or export from the nucleus (4). In prokaryotes, regulatory systems of
replication initiation have been shown to involve fewer regulatory components and are regulated more by the environment than an internal cell cycle. In rich media, for example, cells can
grow with overlapping replication cycles, enabling cell-division times shorter than one round
of genome replication. Here, rigid and global regulation of initiation allows simultaneous
firing on partially replicated chromosomes (5).
Replication of the circular E. coli chromosome is initiated at a single origin of replication, a
245-bp specific sequence called oriC. Key regulator of DNA replication is the AAA+-family
ATPase DnaA that binds to the five 9-bp long DnaA boxes within oriC. Oligomerization of
ATP-bound DnaA along DNA induces positive supercoiling and local melting of the three
nearby located AT-rich 13-bp long sequences called DUE (DNA unwinding element). This
local unwinding then allows binding of two complexes that each consist of the replicative
helicase DnaB and its loader protein DnaC (6-9). The next steps of replication depend on
the concerted action of several key players at the replication fork. The ring-structured replicative helicase DnaB encircles single-stranded DNA and couples the energy released from
nucleotide hydrolysis to directional movement und unwinding of the parental duplex. DNA
polymerase III holoenzyme, a complex of eight different subunit proteins, synthesises the
two daughter DNA strands, starting from RNA primers produced by the primase DnaG (a
detailed description of the bacterial replisome is given in Chapter 2).
Intriguingly, these replisomal machineries cannot be purified as intact complexes - in contrast to for example the macromolecular ribosome (10). The paradoxal situation of a complex
that supports highly processive replication of millions of basepairs while being held together
with weak interaction has been subject of recent research studies (11-13). These studies have
shown that multi-site interactions within the replication complex seem to allow dynamic exchange of DNA polymerases and polymerase holoenzymes while ensuring robustness of the
complex. With the classical picture of a stable replisome recently being challenged, the question arises whether the replicative helicase DnaB remains stably integrated in the replisome
(14, 15) or whether it also dynamically exchanges protein components with those present in
11
Chapter 1
solution. In this thesis, we challenged the stability of DnaB during loading and replication
fork progression.
1.3 DNA sensing by the auto-immune system
Regulation of DNA replication initiation not only impacts the cellular division cycle, but also
presents an important factor in combating invading pathogens and preventing parasitic replication. This process lies at the interface of DNA metabolism and cellular immunity. In both
bacteria and eukaryotes, immune systems have been discovered that either act adaptively
against invading pathogens and build a immunological memory against the respective organism, or that are encoded in the innate immune system, providing a direct and broader
response.
In bacteria, the CRISPR/Cas system has been recently identified to serve as an efficient adaptive immune strategy that memorizes past invasions of bacteriophages (16, 17). An innate
defense mechanism is provided by the restriction-modification system, relying on the expression of restriction enzymes that cleave specific sequences of non-methylated DNA (18).
To protect the self-DNA, DNA methylase enzymes methylate the corresponding sequences
within the genome immediately after replication.
Vertebrates possess highly complex adaptive and innate immune systems to respond to
pathogenic threats. Whereas adaptive immunity is based on clonal gene rearrangements to
express antigen-specific receptors, the innate immune system has been recognized as more
general (19). Innate immune reactions present the first line of defense, with their activation
upon pathogenic infection and cell invasion, and seem to be a prerequisite for activation of
the adaptive immune system (19-21). Several classes of sensors, named Pattern-Recognition
Receptors (PRRs), have been identified that detect structural elements of pathogens, so-called
Pathogen-Associated Molecular Patterns (PAMPs), and that trigger signaling cascades for the
expression of pro-inflammatory molecules. Usually, PAMPs are structural elements such as
nucleic acids, surface glycoproteins, lipoproteins and membrane components of pathogens,
each recognized by specialized PRRs (19). Pathogenic membrane structures such as glycoproteins are recognized by the immune system as foreign, since these structures normally do
not exist inside the eukaryotic host cell. But how does a cell distinguish foreign DNA from
its own genomic material? Different scenarios are possible: 1) an exclusively cytoplasmic localization of the PRR, 2) detection of specific motifs of pathogenic or damaged DNA such as
unmethylated CpG islands, or 3) detection of structural properties of foreign DNA such as
the lack of tight nucleosomal packing (20, 22). The strict discrimination between foreign and
self-DNA is a substantial requirement for the proper activation of inflammatory reactions.
As a consequence, auto-immune disorders often result from misregulation in the process of
foreign-DNA detection or downstream processes (23, 24).
A number of DNA-recognizing PRRs have been reported to be confined to the cytoplasm,
like AIM-2 (25), or to exclusively detect pathogen-specific DNA motifs such as CpG islands,
like TLR-9 (26, 27). Amongst the different human PRRs that detect DNA, the IFI16 protein
is unusual in that it is present within the cytoplasm as well as the nuclear region (28-30).
12
DNA sensing
Cytoplasm
Viruses
STAT6
chemokines
inflammatory
responses
STAT6
NF-κB
Pathogen Associated
Molecular Patterns
Parasites
Sensors
TLR9 IFI16
IFI16
Nucleus
IFI16
AIM2 PolIII
Rig-1 cGAS
ASC
ER
STING
Casp
Figure 2: Pathogen detection by PAMP sensing. Pattern recognition receptors such as Toll-Like-Receptors (TLR’s),
AIM2, and IFI16 have as a key task the detection of certain foreign structures. IFI16 is present in the nucleus, where
it senses pathogen DNA and triggers export of the DNA to the cytoplasm. STING and ASC/caspase-1 are assumed
to interact with the IFI16-DNA complex and start the inflammatory response.
First shown to be engaged in a p53-mediated apoptotic pathway (31, 32), IFI16 is now mostly
thought to be involved in immune mechanisms against invading pathogens, such as Kaposi’s
sarcoma-related herpes virus (KSHV), HIV, listeria or salmonella (28, 33-35). IFI16 belongs
to the interferon (IFN)-inducible p200-protein family, with two 200-amino acid long domains (HIN-A and HIN-B) at the C-terminus that interact sequence-independently with the
sugar-backbone of the DNA (36), and an N-terminal Pyrin domain. It has been demonstrated
that the tandem-arranged HIN domains each bind independently to dsDNA, whereas the Pyrin domain promotes protein-protein interactions (37). An N-terminal nuclear localization
sequence enables transport to the nucleus, with lysine acetylations playing a regulatory role
in the cellular localization (38).
Upon foreign-DNA detection, IFI16 activates an inflammatory response and restricts the replication and transcription of pathogenic DNA in infected cells. Orzalli and co-workers have
shown that IFI16 mainly limits un- or underchromatinized viral DNA, when comparing the
effects on transfected bare SV40 DNA with those seen in SV40 viral infection, with the viral
DNA nucleosomally packaged by host-cell histones (39). After detection of pathogenic DNA,
the IFI16-DNA complex is exported to the cytosol and activates STING (stimulator of interferon genes) (40) and caspase-1 (41). Regulation mechanisms of IFI16-induced inflammatory
responses are not understood in depth, however a cellular antagonist, the Pyrin-domain only
protein (POP3), was shown to inhibit IFI16-mediated processes (42, 43). Together with in
vitro analyses that proved that the Pyd domain is responsible for oligomerization, catalyzed
by binding to DNA (37), inflammatory IFI16 action is assumed to rely on very specific aggregation on foreign DNA. How this detection and aggregation process is achieved, has been one
of our main interests in this thesis.
13
Chapter 1
1.4 Aim of this thesis
As seen from this brief overview, DNA replication, sensing, and control are key biological
processes. In this thesis, using state-of-the-art fluorescence microscopy techniques and data
analysis tools, we aim at unraveling main aspects of DNA metabolism on the single-molecule
level. Real-time, non-invasive techniques such as single-molecule fluorescence imaging offer
insights into transient biochemical steps in DNA interaction or enzymatic cycles and help us
identify intermediates in DNA-protein complex assembly. We profit from classical biochemical techniques that enable the high-throughout generation of protein-DNA interaction maps
(44, 45) and elucidate structural motifs of DNA sequence specificity by proteins by atomic
resolution structures (46, 47), and bulk activity assays to resolve DNA metabolism by specialized enzymes. By observing at the single-molecule level assembly steps of the IFI16 inflammatory complex on DNA, as well as of the replicative DnaB helicase within the replisomal
complex, we can quantify binding stabilities of single molecules versus protein complexes,
characterize DNA-binding modes and search mechanisms along DNA.
In Chapter 2, we summarize biophysical developments that have contributed to our understanding of the fundamental mechanisms underlying DNA replication. We focus on technologies especially in fluorescence microscopy and their applications in single-molecule studies
on different replication systems such as bacteriophage T7, E. coli and eukaryotic cells. We
strengthen the impact of high-resolution techniques to build mechanistic models of multienzymatic and multiprotein reactions en detail, providing spatial and time information of
intermediate reactions and protein interactions that cannot be achieved by ensemble averaging methods.
In Chapter 3, we implement single-molecule tools to characterize the dynamics of the bacterial helicase DnaB at the replication fork. We show that DnaB stably associates at artificial
DNA forks and allows subsequent replisome assembly, thereby supporting efficiently replication initiation. Relative to the lifetimes of all other replisomal components and the discontinuity frequency during coupled replication, we show that DnaB remains stably incorporated
within the replisome and thus may act as the central organizing hub within the replisome to
provide its overall integrity.
In Chapter 4, we use a combined biochemical and biophysical approach to unravel the nucleic-acid detection mechanism of the human DNA sensor IFI16. We show that IFI16 scans
along duplex DNA with a high one-dimensional diffusional velocity to search for other IFI16
molecules, eventually forming a multi-protein complex stably associated with DNA. This
complex serves as signaling platform to trigger inflammatory pathways and is physiologically
switched on upon pathogen invasion into the cellular nucleus. We show that highly specific
sensing of foreign DNA relies on the increased reaction cross section that IFI16 can exploit
on under-chromatinized genomes.
Chapter 5 comprises a methodological advance related to practical challenges encountered
in observing DNA-protein interactions at the single-molecule level. One major hurdle in the
research described in this thesis is the passivation of support surfaces to prevent nonspecific
interactions between these surfaces and the protein-DNA complexes under study. In Chapter 5, we describe the development of a novel microfluidic chip design based on suspended
14
Aim of this thesis
nanowires. We span gold nanowires across channels and apply a straightforward protocol
for functionalization and biomolecule attachment. This configuration allows us to analyze
protein-DNA associations and dynamics even at high protein number per DNA molecule.
We use IFI16 diffusion and aggregation along DNA to demonstrate the advantages of the
suspended nanowire configuration compared to established surface immobilization protocols
in single molecule microscopy.
1.5 References
1.
F. Miescher, Ueber die chemische Zusammensetzung der Eiterzellen. Hoppe-Seyler’s medicinisch-chemische Untersuchungen (Tuebingen, Germany, 1871), vol. 4.
2.
A. Kossel, Ueber einen peptonartigen Bestandtheil des Zellkerns. Hoppe-Seyler’s medicinisch-chemische
Untersuchungen (1884).
3.
K. A. H. Mörner, Nobel Award Ceremony Speech for Prof. A. Kossel. (1910).
4.
S. P. Bell, A. Dutta, DNA replication in eukaryotic cells. Annu Rev Biochem 71, 333 (2002).
5.
K. Skarstad, E. Boye, H. B. Steen, Timing of initiation of chromosome replication in individual Escherichia coli cells. The EMBO journal 5, 1711 (Jul, 1986).
6.
M. J. Davey, D. Jeruzalmi, J. Kuriyan, M. O’Donnell, Motors and switches: AAA+ machines within the
replisome. Nature reviews. Molecular cell biology 3, 826 (Nov, 2002).
7.
J. P. Erzberger, M. M. Pirruccello, J. M. Berger, The structure of bacterial DnaA: implications for general
mechanisms underlying DNA replication initiation. The EMBO journal 21, 4763 (Sep 16, 2002).
8.
K. E. Duderstadt, J. M. Berger, AAA+ ATPases in the initiation of DNA replication. Critical reviews in
biochemistry and molecular biology 43, 163 (May-Jun, 2008).
9.
K. E. Duderstadt, K. Chuang, J. M. Berger, DNA stretching by bacterial initiators promotes replication
origin opening. Nature 478, 209 (Oct 13, 2011).
10.
S. Belin et al., Purification of ribosomes from human cell lines. Current protocols in cell biology / editorial
board, Juan S. Bonifacino ... [et al.] Chapter 3, Unit 3 40 (Dec, 2010).
11.
H. J. Geertsema, A. W. Kulczyk, C. C. Richardson, A. M. van Oijen, Single-molecule studies of polymerase
dynamics and stoichiometry at the bacteriophage T7 replication machinery. Proc Natl Acad Sci U S A 111, 4073 (Mar
18, 2014).
12.
Q. Yuan, P. R. Dohrmann, M. D. Sutton, C. S. McHenry, DNA Polymerase III, but not Polymerase IV,
Must be Bound to tau-Containing DnaX Complex to Enable Exchange into Replication Forks. The Journal of biological chemistry, (Apr 7, 2016).
13.
C. Aberg, K. E. Duderstadt, A. M. van Oijen, Stability versus exchange: a paradox in DNA replication.
Nucleic Acids Res, (Apr 25, 2016).
14.
R. Galletto, M. J. Jezewska, W. Bujalowski, Unzipping mechanism of the double-stranded DNA unwinding by a hexameric helicase: The effect of the 3 ‘ Arm and the stability of the dsDNA on the unwinding activity of the
Escherichia coli DnaB helicase. J. Mol. Biol. 343, 101 (Oct, 2004).
15.
N. Ribeck, D. L. Kaplan, I. Bruck, O. A. Saleh, DnaB helicase activity is modulated by DNA geometry and
force. Biophysical journal 99, 2170 (Oct 6, 2010).
16.
R. Barrangou et al., CRISPR provides acquired resistance against viruses in prokaryotes. Science 315,
1709 (Mar 23, 2007).
17.
M. Goren, I. Yosef, R. Edgar, U. Qimron, The bacterial CRISPR/Cas system as analog of the mammalian
adaptive immune system. RNA biology 9, 549 (May, 2012).
15
Chapter 1
18.
D. Nathans, H. O. Smith, Restriction endonucleases in the analysis and restructuring of dna molecules.
Annu Rev Biochem 44, 273 (1975).
19.
T. H. Mogensen, Pathogen recognition and inflammatory signaling in innate immune defenses. Clinical
microbiology reviews 22, 240 (Apr, 2009).
20.
D. S. Mansur, G. L. Smith, B. J. Ferguson, Intracellular sensing of viral DNA by the innate immune system.
Microbes and infection / Institut Pasteur, (Oct 11, 2014).
21.
A. Iwasaki, R. Medzhitov, Toll-like receptor control of the adaptive immune responses. Nature immunology 5, 987 (Oct, 2004).
22.
H. Ishikawa, G. N. Barber, STING is an endoplasmic reticulum adaptor that facilitates innate immune
signalling. Nature 455, 674 (Oct 2, 2008).
23.
T. K. Means et al., Human lupus autoantibody-DNA complexes activate DCs through cooperation of
CD32 and TLR9. The Journal of clinical investigation 115, 407 (Feb, 2005).
24.
A. Marshak-Rothstein, Toll-like receptors in systemic autoimmune disease. Nature reviews. Immunology
6, 823 (Nov, 2006).
25.
V. Hornung et al., AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome
with ASC. Nature 458, 514 (Mar 26, 2009).
26.
H. Hemmi et al., A Toll-like receptor recognizes bacterial DNA. Nature 408, 740 (Dec 7, 2000).
27.
S. R. Paludan, A. G. Bowie, Immune sensing of DNA. Immunity 38, 870 (May 23, 2013).
28.
N. Kerur et al., IFI16 acts as a nuclear pathogen sensor to induce the inflammasome in response to Kaposi
Sarcoma-associated herpesvirus infection. Cell host & microbe 9, 363 (May 19, 2011).
29.
V. Dell’Oste et al., Innate nuclear sensor IFI16 translocates into the cytoplasm during the early stage of
in vitro human cytomegalovirus infection and is entrapped in the egressing virions during the late stage. Journal of
virology 88, 6970 (Jun, 2014).
30.
S. Costa et al., Redistribution of the nuclear protein IFI16 into the cytoplasm of ultraviolet B-exposed
keratinocytes as a mechanism of autoantigen processing. The British journal of dermatology 164, 282 (Feb, 2011).
31.
R. W. Johnstone, W. Wei, A. Greenway, J. A. Trapani, Functional interaction between p53 and the interferon-inducible nucleoprotein IFI 16. Oncogene 19, 6033 (Dec 7, 2000).
32.
J. A. Aglipay et al., A member of the Pyrin family, IFI16, is a novel BRCA1-associated protein involved in
the p53-mediated apoptosis pathway. Oncogene 22, 8931 (Dec 4, 2003).
33.
K. E. Johnson et al., IFI16 Restricts HSV-1 Replication by Accumulating on the HSV-1 Genome, Repressing HSV-1 Gene Expression, and Directly or Indirectly Modulating Histone Modifications. PLoS pathogens 10,
e1004503 (Nov, 2014).
34.
K. E. Johnson, L. Chikoti, B. Chandran, Herpes simplex virus 1 infection induces activation and subsequent inhibition of the IFI16 and NLRP3 inflammasomes. Journal of virology 87, 5005 (May, 2013).
35.
M. R. Jakobsen et al., IFI16 senses DNA forms of the lentiviral replication cycle and controls HIV-1 replication. Proc Natl Acad Sci U S A 110, E4571 (Nov 26, 2013).
36.
T. Jin et al., Structures of the HIN domain:DNA complexes reveal ligand binding and activation mechanisms of the AIM2 inflammasome and IFI16 receptor. Immunity 36, 561 (Apr 20, 2012).
37.
S. R. Morrone et al., Cooperative assembly of IFI16 filaments on dsDNA provides insights into host defense strategy. Proc Natl Acad Sci U S A 111, E62 (Jan 7, 2014).
38.
T. Li, B. A. Diner, J. Chen, I. M. Cristea, Acetylation modulates cellular distribution and DNA sensing
ability of interferon-inducible protein IFI16. Proc Natl Acad Sci U S A 109, 10558 (Jun 26, 2012).
39.
M. H. Orzalli, S. E. Conwell, C. Berrios, J. A. DeCaprio, D. M. Knipe, Nuclear interferon-inducible protein
16 promotes silencing of herpesviral and transfected DNA. Proc Natl Acad Sci U S A 110, E4492 (Nov 19, 2013).
40.
16
L. Unterholzner et al., IFI16 is an innate immune sensor for intracellular DNA. Nature immunology 11,
References
997 (Nov, 2010).
41.
K. M. Monroe et al., IFI16 DNA sensor is required for death of lymphoid CD4 T cells abortively infected
with HIV. Science 343, 428 (Jan 24, 2014).
42.
S. Khare et al., The PYRIN domain-only protein POP3 inhibits ALR inflammasomes and regulates responses to infection with DNA viruses. Nature immunology 15, 343 (Apr, 2014).
43.
D. J. Connolly, A. G. Bowie, The emerging role of human PYHIN proteins in innate immunity: Implications for health and disease. Biochemical pharmacology, (Sep 6, 2014).
44.
D. S. Johnson, A. Mortazavi, R. M. Myers, B. Wold, Genome-wide mapping of in vivo protein-DNA interactions. Science 316, 1497 (Jun 8, 2007).
45.
in Mapping Protein/DNA Interactions by Cross-Linking. (Institut national de la sante et de la recherche
medicale (INSERM). Paris, 2001).
46.
R. Rohs et al., Origins of specificity in protein-DNA recognition. Annu Rev Biochem 79, 233 (2010).
47.
R. Rohs, S. M. West, P. Liu, B. Honig, Nuance in the double-helix and its role in protein-DNA recognition.
Current opinion in structural biology 19, 171 (Apr, 2009).
17
18