Role of Yeast DNA Polymerase Epsilon During DNA Replication

Role of yeast DNA polymerase epsilon during DNA replication

ISABELLE ISOZ

Akademisk avhandling

Department of Medical Biochemistry and Biophysics UMEÅ UNIVERSITY, SWEDEN UMEÅ 2008 Department of Medical Biochemistry and Biophysics Umeå University

SE-903 87 Umeå, Sweden

New series No. 1231

Printed By: Print & Media, Umeå University, Umeå 2008

2 Table of context

Abstract ...... 5

List of Publications...... 6

1. Introduction ...... 7 DNA ...... 7 DNA polymerases ...... 8 DNA polymerase α...... 10 DNA polymerase δ ...... 11 DNA polymerase ε ...... 12 Replication factors...... 14 Replication protein A (RPA) ...... 14 Replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) ...... 15 Eukaryotic DNA replication ...... 15 Initiation of chromosomal DNA replication...... 15 The DNA replication fork ...... 16 Leading versus lagging strand...... 17 Fidelity...... 19 In vitro ...... 19 Gel assay ...... 19 The gap filling assay ...... 20 In vivo...... 21 Active site...... 22

2. Aims of thesis: ...... 23

3. Results and discussion:...... 24 Paper I ...... 24 Paper II...... 25 Paper III: ...... 26 Paper IV...... 27

4. Concluding remarks...... 29

5. Acknowledgements...... 32

6. References...... 34

3 4 Abstract Each cell division, the nuclear DNA must be replicated efficiently and with high accuracy to avoid mutations which can have an effect on cell function. There are three replicative DNA polymerases essential for the synthesis of DNA during replication in eukaryotic cells. DNA polymerase α (Pol α) synthesize short primers required for DNA polymerase δ (Pol δ) and DNA polymerase ε (Pol ε) to carry out the bulk synthesis. The role of Pol δ and Pol ε at the replication fork has been unclear. The aim of this thesis was to examine what role Pol ε has at the replication fork, compare the biochemical properties of Pol δ and Pol ε, and to study the function of the second largest and essential subunit of Pol ε, Dpb2. To identify where Pol ε replicates DNA in vivo, a strategy was taken where the active site of Pol ε was altered to create a mutator polymerase leaving a unique error-signature. A series of mutant pol ε proteins were purified and analyzed for enzyme activity and fidelity of DNA synthesis. Two mutants, M644F and M644G, exhibited an increased mutation rate and close to normal polymerase activity. One of these, the M644G gave rise to a specific increase of mismatch mutations resulting from T-dTMP mis-pairing during DNA synthesis in vitro. The M644G mutant was introduced in yeast strains carrying a reporter gene, URA3, on either side of an origin in different orientations. Mutations which inactivated the URA3 gene in the M644G mutant strains were analyzed. A strand specific signature was found demonstrating that Pol ε participates in the synthesis of the leading strand. Pol δ and Pol ε are both stimulated by the processivity clamp, PCNA, in in vitro replication assays. To clarify any differences they were challenged side by side in biochemical assays. Pol ε was found to require that single-stranded template (ssDNA) was entirely coated with RPA, whereas Pol δ was much less sensitive to uncoated ssDNA. The processivity of Pol δ was stimulated to a much higher degree by PCNA than of Pol ε. In presence of PCNA the processivity of Pol δ and Pol ε was comparable. In contrast, Pol ε was approximately four times slower than Pol δ when replicating a single-primed circular template in the presence of all accessory proteins and an excess of polymerase. The biochemical characterization of the system suggests that Pol ε and Pol δ are loaded onto the PCNA-primer-ternary complex by separate mechanisms. A model is proposed where the loading of Pol ε onto the leading strand is independent of the PCNA interaction motif which is required by enzymes acting on the lagging strand. The essential gene DPB2 encodes for the second largest subunit of Pol ε. We carried out a genetic screen in S.cerevisiae and isolated a lethal mutant allele of dpb2 (dpb2-200). When over-expressed together with the remaining three subunits of Polε, Pol2, Dpb3 and Dpb4, the dpb2-201 did not copurify. The biochemical property of Pol2/Dpb3/Dpb4 complex was compared with wild-type four-subunit Pol ε (Pol2/Dpb2/Dpb3/Dpb4) and a Pol2/Dpb2 complex in replication assays. The absence of Dpb2 in the complex did not significantly affect the specific activity or the processivity, but gave a slightly reduced efficiency in holoenzyme assays when compared to wild-type four-subunit Pol ε. We propose that Dpb2 is not essential for the enzyme activity of Pol ε.

Keywords: DNA polymerase epsilon, Eukaryotic DNA replication, Fidelity, Leading strand, Dpb2

5 List of Publications

This thesis is based on the following papers:

I. Pursell Z. Isoz I., Lundström EB., Johansson E., Kunkel T.A. Regulation of B family DNA polymerase fidelity by a conserved active site residue: characterization of M644W, M644L and M644F mutants of yeast DNA polymerase ε

Nucleic Acids Res. 2007, 35:3076–3086.

II. Pursell Z. Isoz I., Lundström EB., Johansson E., Kunkel T.A. Yeast DNA polymerase epsilon participates in leading-strand DNA replication.

Science 2007, 317(5834):127-30

III. Chilkova O. Stenlund P., Isoz I., Grabowski P., Lundström EB., Johansson E. The eukaryotic leading and lagging strand DNA polymerases are loaded onto primer-ends via separate mechanisms but have comparable processivity in the presence of PCNA.

Nucleic Acids Res. 2007, 35:6588-97

IV. Isoz I, Volkov K, Johansson E. Characterization of a DNA polymerase epsilon complex lacking the essential subunit Dpb2

Manuscript

6 1. Introduction

Every life form, from a simple virus to a human cell, need to replicate their genome in order to divide. The human genome is built up by 3 billion nucleotides. A simpler eukaryotic single-celled organism, budding yeast, carries about 6,600 genes containing 12 million nucleotides that must be copied with high accuracy in a short amount of time. Mistakes during the replication of DNA may result in mutations that in some cases can cause cell death, cancer, or inheritable diseases in humans. Apart from replication before cell division, DNA must be repaired on a continuous basis in response to damaging agents and conditions such as irradiation with UV light. To protect the genome, a complicated set of mechanisms has evolved.

The process of DNA replication is highly conserved in most organisms. A great deal of information about how DNA replication occurs comes from simple viral systems such as SV40 or single-celled eukaryotic organisms such as budding yeast.

In this work the main focus is on the function of DNA polymerase ε from the budding yeast, Saccharomyces cerevisiae.

DNA

The DNA is built up of four different building blocks, deoxyribonucleotides: deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxycytidine triphosphate (dCTP) and deoxyguanosine triphosphate (dGTP) (reviewed by Nordlund and Reichard, 2006). The nucleotides form specific base pairs between the two strands, to make up the double-stranded DNA helix. The dATP can only pair with dTTP and the dCTP can only pair with dGTP. The double-stranded DNA helix consists of two antiparallel strands running in opposite directions.

During DNA replication, the DNA becomes unwound. The DNA is always synthesized in one direction, 5’ to 3’, giving rise to two different strands: (1) the leading strand, which is replicated continuously, and (2) the lagging strand, which is replicated discontinuously in short segments called Okazaki fragments. (Fig. 1)

Figure 1: The double-stranded DNA helix is unwound, presenting two single stranded templates for the replicative DNA polymerases. The replicative polymerases synthesize new DNA in 5’→3’ direction.

DNA polymerases

The first DNA polymerase was discovered in E. coli at the lab of Arthur Kornberg in the 1950s. In 1970, Wintersberger and Wintersberger published a study in which they had characterized two replicative DNA polymerases from S. cerevisiae (Wintersberger and Wintersberger, 1970).

DNA replication in eukaryotic cells involves 13 different DNA polymerases, of which 8 homologs have been identified in S. cerevisiae, separated into four families based on their primary structure (Table 1). The polymerases responsible for the bulk of DNA synthesis in all eukaryotic cells are DNA polymerase α (Pol α), DNA polymerase δ (Pol δ), and DNA polymerase ε (Pol ε) (reviewed by Garg and Burgers, 2005).

Table 1: DNA polymerases in S.cerevisiae. Catalytic subunits are marked in bold 1(Foury, 1989), 2(Brooke and Dumas, 1991), 3(Burgers and Gerik, 1998),4(Chilkova et al., 2003), 5(Lawrence and Hinkle, 1996), 6(Johnson et al., 1999),7(Baynton et al., 1999), 8(Prasad et al., 1993)

There are no crystallographic data available for the structure of a eukaryotic replicative DNA polymerase. The polymerase most closely related to those of eukaryotic systems is the single-subunit bacteriophage RB69 polymerase. RB69 is to some extent homologous to the eukaryotic replicative polymerases. The structure of this polymerase has been solved to 2.8 Å resolution (reviewed by Wang et al., 1997;Brautigam and Steitz, 1998) The only structural information on a multi-subunit eukaryotic DNA polymerase comes from a low-resolution cryo-EM structure of Pol ε (Asturias et al., 2006). However, there are two recent publications presenting high resolution structures describing a non-catalytic subunit of human Pol δ and a domain of Dpb2, a non-catalytic subunit of human Pol ε (Baranovskiy et al., 2008;Nuutinen et al., 2008)

Comparing the known structures of different DNA polymerases reveals a conserved hand-shaped configuration with a thumb, finger, and palm domain. This conformation has been found in representatives from all the different families (Pelletier et al., 1996;Wang et al., 1997;Doublie et al., 1998;Trincao et al., 2001). Each domain plays an important role in the function of the enzyme.

The DNA polymerase goes through a conformational change during the incorporation of dNTPs into the growing DNA strand. The incoming primer template binds to a groove formed between the palm and thumb domains. This allows the thumb to enfold the DNA by wrapping around the minor groove of the nascent DNA double helix. The incoming dNTP induces a conformational change when the finger domain turns towards the palm, containing the active site, to form a closed binding pocket. This is the polymerization state, when the nucleotide is incorporated into the growing DNA strand (Franklin et al.,

9 2001;Hubscher et al., 2002). The turning of the fingers depends on the insertion of a correct nucleotide. If the incoming nucleotide is wrong, the polymerization rate is reduced, allowing time for the exonuclease domain to remove the mismatch (Franklin et al., 2001;Steitz and Yin, 2004)

By using structural information from the RB69 polymerase and comparing the amino acid sequence at different positions, it is possible to identify homologous sites in other polymerases that can be modified for functional studies.

DNA polymerase α

DNA polymerase α (Pol α) in S. cerevisiae is a monomeric heterotetramer consisting of Pol1p (167 kDa), Pol12p (79 kDa), Pri1p (48 kDa), and Pri2p (62 kDa) (Biswas et al., 2003). The complex has two separate functions: primase and polymerase. Pri1 and Pri2 constitute the primase while Pol1 and Pol12 make up the polymerase (reviewed by Brooke et al., 1991;reviewed by Garg et al., 2005). All subunits are essential for cell viability (Campbell, 1993;reviewed by Foiani et al., 1997).

Pol1 and Pri1 are the catalytic subunits while Pol12 and Pri2 both lack known enzymatic functions (reviewed by Foiani et al., 1997;Garg et al., 2005). The roles of Pol12 and Pri2 are unclear. The Pri12p is phosphorylated during the S phase and may be involved in modulating the role of Pol α primase during the replication (Foiani et al., 1995). Two-hybrid assays have shown that the C terminus of Pol1 is essential for the interaction with Pol12 (Biswas et al., 2003). Early biochemical studies indicated that Pol12 has a role in stabilizing the Pol α complex (Brooke et al., 1991).

During replication, the Pri1 subunit synthesizes an RNA primer of 8–12 nucleotides (nt) after which a switch occurs and Pol1 extends the primer with approximately 20 nt of DNA before Pol α dissociates from the template and is replaced by Pol δ/ε, which continues with the bulk replication (reviewed by Garg et al., 2005).

Pol α lacks exonuclease activity; however, genetic studies have shown that errors by Pol α during DNA replication are corrected by Pol δ (Pavlov et al., 2006a).

10 DNA polymerase δ

DNA polymerase δ (Pol δ) in S. cerevisiae is a monomeric heterotetramer consisting of Pol3p (125 kDa), Pol31p (55 kD), and Pol32p (40 kDa) (Burgers et al., 1998). In humans and fission yeast (Schizosaccharomyces pombe), Pol δ contains an additional small subunit, Cmd1 (fission yeast) or p12 (human), that functions to stabilize the polymerase complex (Zuo et al., 1997;Liu et al., 2000;Podust et al., 2002). The genes POL3 and POL31 in yeast are essential for cell viability, while POL32 is not (Gerik et al., 1998).

Pol3 is the catalytic subunit of the polymerase, and it contains both the exonuclease and polymerase activity domains. The C terminus of Pol3 contains a cysteine-rich region that has been suggested to be part of a zinc-finger domain. Substitution of these cysteines leads to loss of viability (Giot et al., 1997). Studies of the zinc-finger domain on the Pol3 ortholog in fission yeast showed that it is responsible for the interactions with the second subunit, Cdc1, the ortholog of Pol31 (Sanchez et al., 2004).

The subunits of Pol δ are held together by interactions with Pol31 (Burgers et al., 1998). Mutations of conserved sites of the Pol31 subunit have shown that Pol31 is important for the integrity and structure of Pol δ (Vijeh Motlagh et al., 2006).

The smallest subunit, Pol32, is not essential in S. cerevisiae; strains lacking POL32 are cold-sensitive and have an anti-mutator phenotype. The deletion is synthetically lethal in combination with temperature-sensitive alleles of Pol3 or Pol31 (Eissenberg et al., 1997;Gerik et al., 1998). Pol32 is important for interaction with the proliferating cell nuclear antigen (PCNA). The C-terminal end of Pol32 contains a conserved PCNA-interaction motif QXX(L/I)XXFF, a PIP-box; deletion of this motif leads to reduced processivity (Gerik et al., 1998;Johansson et al., 2004). Biochemical studies of PCNA, carrying different mutations that eliminate the interaction with Pol32, have shown that Pol δ can interact with PCNA through other sites positioned on the remaining subunits (Eissenberg et al., 1997;Johansson et al., 2004). The same was shown by deletion of the motif in Pol32 (Johansson et al., 2004). Pol32 has also been found to interact with the catalytic subunit of Pol α: Pol1 (Huang et al., 1999;Johansson et al., 2004).

It has been suggested that Pol δ is involved in the synthesis of both leading and lagging strand (see below). Apart from the replicative function of Pol δ, it also plays an important role in different kinds of DNA repair, such as nucleotide excision repair, mismatch repair, and homologous recombination (Shcherbakova et al., 2003a).

11 DNA polymerase ε

DNA polymerase ε (Pol ε) in S. cerevisiae is a four-subunit complex consisting of Pol2p (256 kDa), Dpb2p (79 kDa), Dpb3p (23 kDa), and Dpb4p (22 kDa) (Hamatake et al., 1990;Chilkova et al., 2003). POL2 and DPB2 are essential for cell viability while DPB3 and DPB4 are not. The subunits of Pol ε can be overexpressed and purified as two separate complexes, Pol2p-Dpb2p and Dpb3p- Dpb4p (Tsubota et al., 2006).

Pol2 is the catalytic subunit of Pol ε, and contains both the polymerase and the exonuclease proofreading activity in the N-terminal domain (Dua et al., 1998). Despite this, it is only the C-terminal half of the subunit that is essential for cell survival (Morrison et al., 1990;Dua et al., 1999;Kesti et al., 1999;Feng and D'Urso, 2001). Cells completely lacking the N-terminal encoding part of POL2 are viable but have impaired growth, while certain point mutations leading to the inactivation of the catalytic site, result to loss of viability. This suggests that another polymerase, presumably Pol δ, can compensate for the lost activity when the N-terminal encoding part of POL2 is missing. (Dua et al., 1999)

Comparison of the amino acid sequences encoded by POL2 from different organisms has revealed several conserved domains in the N-terminal part of the subunit, including the sites responsible for the polymerase and exonuclease activities (Fig. 2). The C terminus contains only two conserved regions: zinc fingers and an ATPase domain, with unknown functions. (Oshige et al., 2000) The two zinc fingers have been suggested to be important for the response to DNA damage at the S-phase checkpoint (Navas et al., 1995;Dua et al., 1998). Deletion of amino acids located between the zinc-finger domains of POL2 leads to loss of viability (Dua et al., 1998). The C-terminal domain is also important for the interaction between Pol2 and Dpb2 (Dua et al., 2000).

Figure 2: Domains of the Pol2 subunit. The putative PCNA interaction motif is located between aa1193-1201 3b shows the cryo-EM structure of Pol ε.

The functions of the three non-catalytic subunits Dpb2, Dpb3, and Dpb4 are unclear. Dpb2 in budding yeast is phosphorylated by cdc28 kinase at multiple

12 positions during late G1 phase. This phosphorylation has been suggested to facilitate the interaction with Pol2 (Kesti et al., 2004). Deletion of Dpb2 in fission yeast (S. pombe) leads to an arrest in early S phase, indicating that Dpb2 may have a role in the initiation of DNA replication (Feng et al., 2003). In a recent paper investigating dpb2 mutant strains in vivo, the authors suggested that Dpb2 in budding yeast contributes to the high fidelity (i.e. accuracy) of Pol ε (Jaszczur et al., 2008).

Dpb3 and Dpb4 together form a complex that has affinity for dsDNA. The ability to bind dsDNA is required for epigenic silencing of telomeres (Tsubota et al., 2006). The nucleotide sequences of the two small subunits contain histone fold motifs (Li et al., 2000) (Ohya et al., 2000). DPB3 is essential for cell viability in fission yeast; cells lacking the gene remain in S phase (Spiga and D'Urso, 2004).

Pol ε has an elongated structure (Chilkova et al., 2003;Asturias et al., 2006). The cryo-EM structure shows that the polymerase is built up of two structural domains: the head domain consisting of Pol2, and the tail domain consisting of Dpb2, Dpb3, and Dpb4 (Fig. 3). The tail domain has been shown to be important for the high processivity of the polymerase (Asturias et al., 2006).

Figure 3: Cryo-EM Structure of Pol ε, Pol2 constitutes the head domain while Dpb2/Dpb3/Dpb4 makes up the flexible taildomain (Asturias et al., 2006).

Early on in Pol ε research, its high processivity in combination with its high fidelity led to the suggestion that Pol ε is involved in DNA replication (Morrison et al., 1990). Pol ε possesses the 3’-5’ exonuclease activity that is also found in Pol δ. Inactivation of the exonuclease activity in vivo, result in mutator phenotypes (Morrison et al., 1991;Simon et al., 1991;Morrison and Sugino, 1994).

13 Pol ε was initially identified in HeLa cells as a form of Pol δ not stimulated by PCNA (Syvaoja and Linn, 1989). In S. cerevisiae, a homolog of this polymerase was characterizeed under the name of DNA polymerase II (Hamatake et al., 1990;Morrison et al., 1990). The catalytic subunit, Pol2, contains a PCNA interaction motif (Dua et al., 1998) and DNA synthesis by Pol ε has been found to be stimulated by PCNA and RFC in holoenzyme assays, but to a lesser degree than Pol δ (Hamatake et al., 1990;Burgers, 1991;Dua et al., 2002;reviewed by Garg et al., 2005). Deletion of this motif was not found to affect cell viability, but led to an increased sensitivity to methyl methanesulfonate (MMS) (Dua et al., 2002).

Pol ε was first identified as a polymerase essential for DNA replication in S. cerevisiae by (Morrison et al., 1990). Since then, it has been suggested to have several roles during the course of the cell cycle. Chromatin immunoprecipitation assays (ChIP assays) have shown that Pol ε is loaded at the origin of replication, both in early and late S phase, indicating that it plays some parts during the initiation of DNA replication (Aparicio et al., 1999). Pol ε interacts with several components that are involved in the initiation of DNA replication, such as GINS, Dpb11, and Cdc45 and has been suggested to be important for the loading of Pol α (Masumoto et al., 2000;Takayama et al., 2003;Shikata et al., 2006). Pol α and Pol ε are loaded at the origin of replication earlier than Pol δ (Hiraga et al., 2005).

Except for the replicative role, Pol ε is an important factor in DNA repair mechanisms. It has been shown to be involved in homologous recombination, mismatch repair, and nucleotide excision repair (Shcherbakova et al., 2003a)

Replication factors

In addition to the DNA polymerases, a large number of accessory proteins are needed to successfully replicate the DNA in vivo (Hubscher and Seo, 2001;reviewed by Johnson and O'Donnell, 2005). Three well-characterized replicative factors are RPA, RFC, and PCNA.

Replication protein A (RPA)

Unwinding of double-stranded DNA by the helicase results in two separate DNA strands. To protect the single-stranded DNA (ssDNA) from nucleases and to avoid formation of hairpin structures that could have a negative effect on successful replication, the ssDNA is coated with an ssDNA binding protein, RPA (Brill and Stillman, 1991;Waga and Stillman, 1998;Fanning et al., 2006). The

14 structure of RPA is conserved in eukaryotic organisms. It consists of three essential subunits, Rpa1p (70 kDa), Rpa2p (30 kDa), and Rpa3p (14 kDa) (Wold, 1997). RPA has been shown to play an important role in stabilizing Pol ε at the primer end on the template (Maki et al., 1998).

Replication factor C (RFC) and proliferating cell nuclear antigen (PCNA)

Replication factor C (RFC) is a protein responsible for loading the clamp, proliferating cell nuclear antigen (PCNA), to the primed template during DNA replication. PCNA tethers Pol ε and Pol δ to the DNA during replication and is responsible for sustained processive replication (Maga and Hubscher, 1995), (Gomes and Burgers, 2001;Gomes et al., 2001;Yao et al., 2006).

RFC in S. cerevisiae is a 5-subunit complex consisting of Rfc1p (95 kDa), Rfc2p (40 kDa), Rfc3p (38 kDa), Rfc4p (36 kDa), and Rfc5p (40 kDa) (Johnson et al., 2005). PCNA is a complex consisting of three identical subunits of 29 kDa that form a ring-like structure that encircles the DNA (Bauer and Burgers, 1990;Krishna et al., 1994).

Eukaryotic DNA replication

DNA replication in eukaryotes is a complex, highly regulated process involving many different replication factors.

Initiation of chromosomal DNA replication

The different proteins involved in the initiation of DNA replication vary between organisms (Kearsey and Cotterill, 2003). The molecular events that take place during these steps are not completely understood. Below follows a simplified description of how chromosomal DNA replication is initiated in S. cerevisiae.

The replication is initiated during G1 phase by ATP-dependent binding of the origin recognition complex (ORC) to origins in the S. cerevisiae genome called autonomously replication sequences (ARSs). The replication is initiated simultaneously at multiple origins, but only once per cell cycle. The binding of the ORC to ARSs leads to the recruitment of Cdc6, which is responsible for loading Cdt1 and the helicase Mcm2-7 (MCM), which together form the pre-

15 replicative complex (pre-RC). The loading of the pre-RC onto an ARS is regulated by ATP-mediated hydrolysis of Cdc6 and ORC (Bowers et al., 2004;Kawasaki et al., 2006;reviewed by Chesnokov, 2007).

As the cells enter S phase, the MCM, Sld2, and Sld3 are phosphorylated by the Cdc7-Dbf4 and the cyclin-dependent kinase CDK. The phosporylation of Sld2 and Sld3 are essential for the binding of Dpb11, which allows recruitment of GINS, Cdc45, and Pol ε to the origin (Kamimura et al., 1998;Kamimura et al., 2001;Takayama et al., 2003;Zegerman and Diffley, 2007;Tanaka et al., 2007). The recruitment of GINS and Cdc45 to the origin results in unwinding of the DNA and binding of RPA to the single-stranded DNA. Phosphorylation of MCM leads to the recruitment of Mcm10, which—in the presence of RPA—has been suggested to play a role in loading Pol α onto the chromatin and stabilizing it (Ricke and Bielinsky, 2004). During the final step of the initiation, RFC loads PCNA onto the newly synthesized primer and recruits Pol δ or Pol ε.

The DNA replication fork

Figure 4: A simplified model of the replication fork in S.cerevisiae

16 The chromosomal replication of the leading strand and the lagging strand is initiated by Pol α. Pol α synthesizes a 20–30 nt RNA/DNA hybrid primer; when this primer has been produced, a switch occurs whereby the RFC binds to the 3’- end of the primer, causing Pol α to dissociate from the template. RFC interacts with PCNA, and through hydrolysis with ATP it opens up the circular PCNA and loads it onto the DNA, recruiting the polymerases responsible for the elongation: Pol δ and Pol ε (Waga et al., 1994;reviewed by Hubscher et al., 2002). (Fig. 4)

The lagging strand in S. cerevisiae is replicated in stretches of 150–200 nt, the Okazaki fragments. When the Okazaki fragment comes to an end—i.e. the polymerase meets a primer originating from a previously replicated fragment—2 to 3 nt are displaced from the downstream primer, creating a 5’-flap containing the RNA. The flap is continuously removed by flap endonuclease (FEN1) leaving a nick in the DNA. The RNA primer must be removed before completion of the lagging strand. The nick is finally sealed by DNA ligase I to give a continuous DNA strand. The entire process is called Okazaki fragment maturation (Garg et al., 2004;Stewart et al., 2006).

Leading versus lagging strand

Figure 5: Three different models describing which DNA polymerase replicates the leading and laggingstrand has been suggested over the years: a) Pol δ replicates both strands. b) Pol δ replicates the leading strand, Pol ε replicates the lagging strand. c) Pol δ replicates the lagging strand, Pol ε replicates the leading strand

The organization of the DNA replication fork has been debated over the years. Due to the primase function of Pol α, early on this polymerase was suggested to be responsible for initiating replication by producing the RNA/DNA hybrid primer essential for DNA replication in eukaryotic cells (reviewed by Foiani et al., 1997).

17 Early studies of the eukaryotic replication fork were done using cell-free extracts infected with plasmids containing an origin from a simian polyoma virus, SV40. The circular genome of SV40 virus consists of about 5,000 base pairs and has one origin for initiation of replication. By only bringing in one gene essential for replicating the DNA, that encoding the T-antigen, the virus genome finds all other factors needed for replication inside the host cell. This made it a good model for studies of eukaryotic replication in vitro (Li and Kelly, 1984;reviewed by Kelly, 1988). Reconstitution of the SV40 replication fork in vitro showed that Pol α, Pol δ, RPA, RFC, PCNA, FEN1, DNA ligase I, and DNA topoisomerases I and II are required to replicate the viral genome (reviewed by Kelly, 1988;Waga et al., 1994;Waga and Stillman, 1994).

Studies of the SV40 system, where Pol ε was not required, gave rise to a model of the replication fork where Pol δ was responsible for the elongation of both the leading strand and the lagging strand (Fig. 5a) (Zlotkin et al., 1996).

Additional studies have been carried out to try to elucidate the separate roles of Pol δ and Pol ε at the replication fork. The identification of the exonuclease sites in Pol ε and Pol δ made it possible to remove the proofreading activity from these polymerases in vivo and in vitro. By studying the effects in exonuclease-deficient yeast strains, it was concluded that the two polymerases proofread different strands (Shcherbakova and Pavlov, 1996;Karthikeyan et al., 2000).

A model positioning Pol δ on the leading strand and Pol ε on the lagging strand (Fig. 5b) was suggested based on assays showing that yeast Pol ε does not form a stable complex with the template in the presence of RPA, PCNA, and ATP (Burgers, 1991). This was later supported by a study mimicking the Okazaki fragment gaps, showing that calf thymus Pol ε but not Pol δ had the ability to replicate a 230-nt gap on an M13 template DNA in the absence of RFC, PCNA, and RPA (Podust and Hubscher, 1993).

When Pol ε and Pol δ were identified as separate polymerases, Pol ε was found to be less dependent on the presence of PCNA than Pol δ (Syvaoja et al., 1989;Hamatake et al., 1990). Mammalian Pol δ had previously been shown to be stimulated by PCNA (Burgers, 1988). Later studies have shown that the two polymerases interact differently with PCNA (Eissenberg et al., 1997;reviewed by Garg et al., 2005). The dependence on PCNA for processivity has been proposed to be a factor characteristic of a lagging-strand polymerase (Garg et al., 2005).

The recent finding that only Pol δ, but not Pol ε, has the ability to idle—i.e. is involved in Okazaki fragment maturation—appears to represent an important function for a lagging-strand polymerase (Jin et al., 2001;Garg et al., 2004). It has also been shown that Pol δ (but not Pol ε) has the ability to correct mistakes made by Pol α during synthesis of the primer (Pavlov et al., 2006a).

In 2005, two reviews were published—positioning Pol δ and Pol ε on opposite strands (reviewed by Garg et al., 2005;reviewed by Johnson et al., 2005). This shows that although a great deal of data place Pol δ on the lagging strand and Pol ε on the leading strand (Fig. 5c), there is still some disagreement in the field and more research will be required to ultimately identify the organization of the replication fork.

Fidelity

Fidelity is a measure of the rate by which mistakes are made by a polymerase during DNA replication. A polymerase of high fidelity causes few mutations. Not all polymerases need to have high fidelity, but mistakes caused by polymerases of low fidelity can in some cases be corrected by a replicative polymerase with exonuclease activity. An important example of this is when Pol δ proofreads the primer produced by Pol α during the replication of the Okazaki fragments (Pavlov et al., 2006a).

To be able to correct misinserted bases, as mentioned above, some DNA polymerases—e.g. Pol δ and Pol ε—possess an intrinsic 3’-5’ exonuclease activity. The amino acid sequence of the exonuclease site, FDIEX, is conserved in different polymerases. It is possible to inactivate the exonuclease activity by substituting the asparagine (D) and phenylalanine (F) by alanines (A), and the inactivation results in a significant increase in the error rate of the polymerase. In both Pol δ and Pol ε, the exonuclease site is positioned at the N-terminal part of the catalytic subunit (Blanco et al., 1992;Morrison et al., 1994).

In vitro

Two methods have been used to study the fidelity of a purified polymerase: a gel-based primer extension assay and a gap-filling assay developed by Thomas A Kunkel whereby the extended template is inserted into E.coli cells.

Gel assay

Primer-extension assays can be used to calculate the correct or incorrect insertion efficiency of a polymerase. A radiolabeled primer-template is incubated with the polymerase and nucleotides. The sample is separated on a denaturating polyacrylamide gel and the intensities of the bands are quantified using

19 phosphoimaging software. The values obtained are then used to estimate the misincorporation efficiency, finc.(Creighton et al., 1995;reviewed by Pavlov et al., 2006b)

By using this method to calculate the misincorporation efficiency of Pol δ and Pol ε from S. cerevisiae, it was found that the exonuclease activity in Pol ε is very efficient. The values increased 100 times compared to the wild-type polymerase when the exonuclease activity was inactivated. Pol δ had higher misincorporation efficiency and the influence of exonuclease activity was only 10-fold (Shimizu et al., 2002;Hashimoto et al., 2003).

The gap filling assay

Another method used to study the fidelity in vitro is the gap-filling assay. This method gives more information on the different mutations caused by the polymerase. Using this method, it is possible to identify a variety of mutations: substitutions, additions, and deletions of one or more bases.

Figure 6: Gap filling assay (Drawing is based on experimental outline from (Creighton et al., 1995)

20 The polymerase is added to a reaction containing a gapped M13mp2 phage DNA and dNTPs. The single-stranded gap contains a lacZ reporter gene. The replicated DNA is inserted into competent E. coli cells plated on an agar plate containing the chromogenic indicator X-gal and a lawn of E. coli cells lacking β- galactosidase activity. If the gap was replicated without errors in the in vitro reaction with the studied DNA polymerase, the resulting plaques develop a dark blue color while cells containing mutated gaps give rise to plaques that are light blue or colorless. The plates are scored for mutation frequency, and the DNA from the mutants is purified; then the 125 bases of the replicated gap are sequenced (Creighton et al., 1995). (Fig. 6)

Both wild-type (wt) Pol δ and wt Pol ε have low error rates in these assays. Pol ε produced less than 2 base substitutions and 0.05 frameshift mutations per 10,000 inserted nucleotides. When the exonuclease site was inactivated, the error rate increased 10 and 100 times, respectively (Shcherbakova et al., 2003b). For Pol δ, the same rates were 1.3 and 1.5 for the wt polymerase and the increase in error rate when the exonuclease activity was removed was 10-fold and 5-fold, respectively (Fortune et al., 2005).

In vivo

In vivo assays are often used as a complement to the in vitro fidelity assays, to obtain a better understanding of the true consequences of a mutated gene.

A type of assay often used for this purpose is the fluctuation test. This method is based on yeast strains containing different marker genes. By inserting the mutation of interest into these cells, it is possible to identify a number of mutations causing errors in DNA replication or proofreading. The selective medium is chosen based on the type of mutation under study and the marker gene present in the cell. For instance, a cell containing a his7-2 gene can be used for identification of mutations resulting in +1 insertion or –2 deletions. Cells that grow on plates lacking histidine in the medium contain reversion mutations in this gene. By simultaneously plating cells on rich medium, it is possible to calculate the mutation frequency in the cells. Other markers that are commonly used are: e.g. trp1-1 and ade2-1, identifying base-pair substitutions, while the CAN1 can be used to screen for any mutations. Assays studying mutations in the his7-2, trp1-1, and ade2-1 are specified as reversion assays while studies of CAN1 mutants are called forward mutation assays. The genetic marker genes are often sequenced to identify the specific mutations caused by the initially mutated gene into the strain (Shcherbakova and Kunkel, 1999;Pavlov et al., 2001;Pavlov et al., 2006a).

21 Active site

Comparison of nucleotide sequences of different DNA polymerases reveals a number of highly conserved regions, called motifs. Three of these are positioned close to, or at, the active site of the polymerase (Zhang et al., 1991;Brautigam et al., 1998;Pavlov et al., 2006b).

The active site of DNA polymerases is located in the palm domain. By studying the structures arising from crystallized polymerases (from RB69, T7, and T4) on a structural/functional level, it has been possible to determine that motifs II and III are positioned at the dNTP binding pocket, which is important for selection of the base to be inserted (Pavlov et al., 2001).

Several different polymerases have been studied in an attempt to gain a better understanding of the function of base selectivity, and to clarify the functional differences and similarities between DNA polymerases. In an early study using the T4 polymerase, a number of mutants were constructed that contained a base substitution at different positions of motif II. This study showed that point mutations at this site could either increase or reduce the fidelity of the polymerase in vitro (Reha-Krantz and Nonay, 1994).

In an attempt to study the effects of mutations in the palm domain of Pol α in S. cerevisiae, a screen was performed to identify mutants containing single substitutions of L868, one of the conserved amino acids in motif II. The consequences of these substitutions were studied both in vitro and in vivo. The mutants identified showed reduced fidelity in vitro and mutator phenotypes in vivo (Niimi et al., 2004).

The effects of mutations at the active site can be used to obtain a better understanding of interactions between different polymerases in vivo.

22 2. Aims of thesis:

The aim of this thesis was to define the role of yeast DNA polymerase ε during DNA replication by studying the polymerase in different context in vitro and in vivo.

Specific aims • To develop mutator alleles of Pol ε, to be used in in vivo studies

• To identify which of the two strands is synthesized by Pol ε during DNA replication

• To compare the Pol ε-PCNA interaction with the Pol δ-PCNA interaction

• To investigate the functional role of Dpb2 in yeast.

23 3. Results and discussion:

Paper I:

Regulation of B family DNA polymerase fidelity by a conserved active site residue: characterization of M644W, M644L and M644F mutants of yeast DNA polymerase ε.

In this paper, we wanted to construct mutants of Pol ε containing an amino acid substitution at the active site and to study the effects of these mutations on fidelity.

We chose to focus on amino acid 644, a methionine (M). This amino acid is situated in a highly conserved part of the active site, called motif II. Substitutions of the amino acid at the position corresponding to M644 in Pol α—L868 (Niimi et al., 2004)—resulted in mutants with reduced fidelity but normal polymerase activity. Our first aim was to determine whether M644 plays the same role in Pol ε as L868 in Pol α and L612 in Pol δ regarding the nucleotide insertion specificity.

Pol ε is a 379-kDa large complex consisting of 4 subunits, and to overexpress and purify this enzyme is a task that takes a lot of time and effort. We intended to construct and study a number of mutants of Pol ε. Thus, to reduce the amount of work required to obtain the mutants, we wanted to determine whether it was possible to use an N-terminal 152-kDa fragment of Pol2 containing the catalytic sites of Pol ε as a model in our in vitro assays. It had already been shown that all the catalytic activity of Pol ε is located in the N-terminal part of its largest subunit, Pol2 (Budd et al., 1989;Dua et al., 1999).

The purified fragment was compared to the 4-subunit complex regarding specific activity and fidelity. To study the fidelity, we used the gap-filling assay whereby the polymerase replicates a single-stranded gap on an M13mp2 DNA. The results showed that neither the specific activity nor the fidelity differed significantly between the 152-kDa fragment and the 4-subunit Pol ε. This confirmed that it was possible to use this fragment as a model for Pol ε in our assays.

In an effort to study the importance of M644 for the fidelity of Pol ε, we constructed three different mutants with M644 substituted by tryptophan (M644W), leucine (M644L), or phenylalanine (M644F).

The initial fidelity measurements were run on exonuclease-deficient mutants compared with the exonuclease deficient wt fragment (M644Mexo-). The M644Wexo- and M644Lexo- mutants showed fidelity comparable to the

24 M644Mexo-. The M644Fexo- was 6.9 times less accurate than the wild type. With sustained proofreading activity, the M644F showed a 2.7-fold higher mutation frequency than the wild-type fragment.

Looking at the specific mutations caused by M644Fexo-, we found that the proportion of misincorporations was increased for all possible errors compared to the M644Mexo-. With sustained proofreading activity, a large number of the single-base substitutions were/was corrected in the M644F mutant.

We succeeded in constructing an active site mutant of Pol ε with reduced fidelity but sustained activity. The results of this study show that the M644 position plays a role in nucleotide insertion specificity, as has been suggested for studies of the corresponding position in other polymerases (Reha-Krantz et al., 1994;Niimi et al., 2004).

Paper II:

Yeast DNA polymerase ε participates in leading-strand DNA replication.

In several previous studies the authors have tried to identify which strand of the replication fork is replicated by Pol ε in vivo (Burgers, 1998); (Garg et al., 2004), (Pavlov et al., 2006a). Using the experience gained from our previous paper, we hoped to find a new mutant that would help us come closer to the answer.

When comparing the fidelity of the M644Gexo- mutant, we found that it had an 8.8-fold higher mutation rate than the M644Mexo-. Looking at the specific errors made by the mutant with sustained proofreading activity, an interesting observation was made. The M644G was especially prone to making T-T mismatches, with a 22-fold difference between the exonuclease prone wild type, the M644M fragment, and the exonuclease-prone M644G. When we compared T-T mismatches to the reciprocal A-A mismatches for the M644G, we saw an almost 40-fold difference. This was confirmed in assays calculating the catalytic efficiency, showing that the M644Gexo- had a much lower Km value for misinsertion of a T-T compared to the M644Mexo-. The rate of T-T mismatches made by the M644G mutant was significantly higher than the rate for Pol δ.

Before inserting a mutation into a yeast strain, it is important to analyze the enzyme at different levels to ensure that the substitution does not cause any additional effects other than the increase in mutation rate, to avoid the risk of involvement of other intracellular mechanisms. We tested the specific activity, the exonuclease activity, and effect on growth rate and found that the mutant functioned normally in these assays. The M644G mutation was inserted into the chromosome of 6 different yeast strains, each containing a URA3 reporter gene positioned in different origins and orientations. The corresponding strains containing the wt Pol ε were constructed. The mutation rates in all strains were

25 compared; there was an increase in mutation rate in the M644G mutant strains compared to the wt. When the sequence of the URA3 gene from these strains was analyzed, it was possible to identify two hotspots with an especially high increase in the rate of T-T mismatches made by the M644G mutant. These hotspots were only found in the M644G mutant strains with the URA3 gene in the leading- strand direction. The occurrence of T-T mismatches was up to 30-fold higher in these strains.

Based on these results, we suggest that Pol ε participates in the synthesis of the leading strand DNA in Saccharomyces cerevisiae.

Paper III:

The eukaryotic leading- and lagging-strand DNA polymerases are loaded onto primer-ends by separate mechanisms, but have comparable processivity in the presence of PCNA.

To clarify any functional differences between Pol δ and Pol ε, we challenged the two polymerases in different assays and studied their affinities for DNA, RPA, and PCNA.

Using holoenzyme assays containing recombinantly overexpressed and purified replication factors RPA, PCNA, and RFC on two different lengths of ssDNA, we could see that Pol δ replicates single-primed circular DNA at least four times faster than Pol ε. The amount of RPA added to the reaction played an important role in the ability of Pol ε to be stimulated by PCNA. Using a low amount RPA, enough to coat only 20% of the single-stranded template DNA, did not result in any visible products on the agarose gel. For successful replication, all the ssDNA had to be coated with RPA. Pol δ showed no sensitivity for ssDNA but was stimulated by PCNA in the presence of both low and high concentrations of RPA.

We wanted to study the true processivity of the two polymerases in the presence of replication factors. To do this, we used a 1:30 ratio of polymerase to primed template and excess of accessory replication factors. This ratio ensures that each polymerase only encounters the template once; when it disassociates from the template, it will not encounter an already extended template. When the ssDNA was partially coated with RPA and no PCNA or RFC was added to the reaction, Pol ε was 10 times more processive than Pol δ. Addition of PCNA and RFC to the reaction had a much greater effect for Pol δ (100-fold) than Pol ε (6-fold). Adding enough RPA to fully coat the ssDNA had only minor effects, for both polymerases.

26 Using the surface plasmon resonance technique, Biacore, we found that Pol ε had high affinity for all the different types of DNA tested (single-stranded, primed, and double-stranded), while Pol δ had no affinity for any of the three DNA types. Immobilization of RPA on the DNA resulted in reduced affinity of Pol ε for single-stranded DNA but a higher affinity for primed DNA.

We also found that Pol ε had no ability to interact with PCNA immobilized on a chip, while Pol δ interacted strongly.

To study how efficiently the PCNA is loaded onto a primer-template in the presence of Pol ε or Pol δ, we used radiolabeled PCNA to measure how many molecules were loaded onto a circular primed template during replication. We found that twice the number of PCNA molecules is loaded by RFC during DNA synthesis with Pol ε than with Pol δ. Finally, we measured the influence of the increased level of PCNA or RFC on the polymerase activity of Pol ε. We could see that increased amounts of PCNA had a positive effect while the RFC concentration did not affect the activity.

We suggest in this paper that Pol ε and Pol δ have similar processivity but are loaded onto the template ends via different mechanism. A model is proposed where the loading of Pol ε onto the leading strand is independent of the PCNA interaction motif which is required by enzymes acting on the lagging strand.

Paper IV:

Characterization of a Pol ε complex lacking the essential subunit Dpb2

In this paper we study the essential role of the second largest subunit of Pol ε, Dpb2.

To identify lethal mutations in the DPB2 gene we constructed a yeast strain, E134-dpb2Δ, with a yeast centromeric vector expressing DPB2 under its own promoter containing a URA3 selectable marker.

To produce mutants of DPB2 we constructed a new vector containing a TRP1- selectable marker. This vector was exposed to a mutative agent, hydroxylamine. The treated vector was inserted into the E134-dpb2Δ strain and the wt-vector was removed by plating the cells on 5-flouroorotic acid, (5-FOA) medium. The 5- FOA medium is toxic to cells containing the URA3-gene. Only cells which can survive without the wt-vector will grow on 5-FOA containing medium.

27 We identified 11 colonies containing lethal mutations in the mutated DPB2-gene. One of these mutants contained 2 substitutions close to the C-terminus of DPB2 (dpb2-200).

To identify whether one of the two substitutions were solely responsible for the lethality, new strains were constructed, dpb2-202 and dpb2-203. The result showed that both substitutions were needed for a lethal phenotype. There was no increase in sensitivity for UV but a slight growth inhibition of the dpb2-202 mutant was seen in presence of HU compared to wt-cells. FACS analysis showed that the dpb2-202 mutation caused a shift in distribution of cells over the cell cycle. We also found that the growth rate of dpb2-202 cells was slower than wt and dpb2-203 cells.

We asked whether the lethal mutation have any affect on the polymerase activity DNA polymerase ε in vitro. For overexpression the remaining amino acids after the stop-codon were removed, to create dpb2-201. When overexpressed together with the other subunits of Pol ε, Pol2, Dpb3 and Dpb4, the dpb2-201 did not copurify with the complex. Western blots showed that the mutant protein was overexpressed. The biochemical properties of this new complex containing Pol2/Dpb3/Dpb4 was compared with the four subunit Pol ε. The specific activity was not significantly affected when lacking Dpb2 in the complex. Next we found that the processivity of Pol ε and the Pol2/Dpb3/Dpb4 complex was comparable. Finally we found that Pol ε needed 4 minutes to fully replicate the 3 kb pBluescript SK II(+) DNA while Pol2/Dpb3/Dpb4 complex needed 8 minutes in holoenzyme assays with all accessory proteins..

Based on the results in our holoenzyme and processivity assays with the four subunits Pol ε and the Pol2/Dpb3/Dpb4 complex we suggest that the lethality in dpb2Δ strains is not due to an inactive DNA polymerase function.

28 4. Concluding remarks

In the first two papers we studied the effects of active site mutations in Pol2. In Paper I we concluded that by substituting an amino acid at the active site, M644, it was possible to reduce the fidelity of the polymerase. In Paper II we found a mutant that gave a specific error signature that could be used to explore the role of DNA polymerase ε at the replication fork.

In the introduction to this thesis the different ideas raised over the years about the organization of the chromosomal DNA replication fork were discussed. In recent years the suggestion that Pol ε is responsible for replicating the leading strand and Pol δ the lagging strand has gained more and more support from different studies (Garg et al., 2004;reviewed by Garg et al., 2005;Pavlov et al., 2006a). However no consensus was established within the field as shown by two reviews published in 2005 giving different suggestions on the composition of the replication fork (reviewed by Garg et al., 2005;reviewed by Johnson et al., 2005).

We showed that Pol ε participates in the synthesis of the leading strand in S.cerevisiae. By using different origins and directions we found that Pol ε is involved in leading strand synthesis in both early and late S-phase. Our data was confirmed by a study published earlier this year where the same approach was used to identify Pol δ as the lagging strand polymerase (Nick McElhinny et al., 2008). This study demonstrated that Pol ε is responsible for most of the synthesis on the leading strand and Pol δ on the lagging strand at the studied regions of the genome.

In paper III we compared the biochemical properties of Pol ε and Pol δ in different assays. The main conclusion from this study is that Pol ε and Pol δ have comparable processivity but are loaded onto the primer end via different mechanisms

The stimulatory effect of PCNA has been suggested to be important for a lagging strand polymerase (reviewed by Garg et al., 2005). A leading strand polymerase is presumably loaded only once during DNA replication, a lagging strand polymerase may need to be loaded fast and efficiently to replicate the large number of Okazaki-fragments.

The results from this paper can in part explain why Pol δ predominantly synthesizes the DNA on the lagging strand. However it does not explain why Pol ε predominantly synthesizes DNA on the leading strand. We hypothesize that

29 there may be a loading mechanism for the leading strand polymerase, which is independent of PCNA.

In Paper IV we studied the function of Dpb2 during replication. We identified a lethal mutant containing two substitutions close to the C-terminus of the gene. Overexpression of the mutant Dpb2 together with the other subunits of Pol ε resulted in a three subunit complex consisting of only Pol2/Dpb3/Dpb4. The comparison of the biochemical properties between the Pol2/Dpb3/Dpb4 complex and the four subunit Pol ε did not reveal any significant differences in activity and processivity. The role of Dpb2 is not clear. Previous studies have suggested that the subunit may be involved in different mechanisms during initiation of DNA replication and contribute to the fidelity of Pol ε during the replication (Araki et al., 1992;Feng et al., 2003;Jaszczur et al., 2008).

The results from our replication assays do not add any conclusive explanation to the enzymatic role of Dpb2. The reduced activity in holoenzyme assays is unlikely to be significant enough to suggest an essential role in DNA polymerase ε during DNA replication.

30 31 5. Acknowledgements

Under de drygt 11 år som har gått sedan jag satte min fot i Ume har jag hunnit träffa en massa underbara människor som kommit att betyda mycket för mig genom åren. Listan skulle bli lång om man skulle tacka var och en. Men jag gör ett försök att nämna några:

To start with: I would like to thank all All the wonderful people on the Department of Medical Biochemistry and Biophysics. The atmosphere of science and pleasure makes the work much easier. You will always have a special place in my heart:

Min handledare Erik Johansson: Jag hade inte kunnat önska mig en bättre handledare. För att du delat med dig av din kunskap och vetgirighet. Tack för att du hjälpt mig att nå hela vägen och aldrig helt gett upp.

The EJ-Group Else-Britt: Vad vore gruppen utan dig som höll ordning på oss? Tack för att du är en sådan klippa och vän. Nasim: För allt roligt prat vid labbänken genom åren, tack för hjälpen med skrivandet. Lycka till på ditt nya äventyr USA! Kirill: For your great knowledge and for always taking time to answer stupid questions about Computers and Science. Anja: for your knowledge and help in the lab. I will miss you all!

Tidigare medlemmar i gruppen: Olga C: Du var den som ledde vägen för oss andra på labbet, Lycka till i Stockholm. Göran B: För att du är den du är.

Our collaborators: Special thanks to Thomas Kunkel and Zachary Pursell: for letting me be part of great Science. Also thanks to Peter Burgers.

Alla som håller institutionen gående: Ingrid och Clas, för att ni alltid håller koll på allt. Elisabeth: för att du alltid har ett leende till övers. Anne-Marie: hoppas att du trivs med din ledighet. Lola: Tack för alla lösningar och plattor, Krya på dig! Urban: Tack för att du höll koll på kurslabbarna.

Alla runt Fika och Lunch, ingen nämnd ingen glömd. Många diskussioner har det blivit genom åren, om allt mellan himmel och jord, men framför allt: Om ett gott skratt förlänger livet, då kommer jag att Leva Läänge!

Anna E, Miriam, Jenny: Tack för att ni alltid har funnits där när allt känts tungt men framför allt för allt prat om annat Kul som hör livet till.

Rima: for discussions about our new experiences, good luck with the baby.

32 Miriam och Kristina: För träning med skidor och löpning, och inte att förglömma våra roadtrips på vägarna mot Mora och Stockholm. Jag gör gärna ett nytt försök om ett par år. Halvklassiker kanske?

Tobias Tengel: HV71 Rules!

- Mången nämnd Ingen glömd! Jag kommer att sakna er alla!

Vännerna utanför labbet:

Anna A: Tack för att du lurade mig att börja jobba på Caelum. Det var början på en vänskap som har lett till mycket skvaller, många skratt, filmer och en hel del annat.

Susanne: Vi har följts åt sedan mitt Ume-äventyr tog sin början. Tack för alla djupa diskussioner om allt och inget, för alla skratt. Tack för hjälpen med skrivandet.

Anna A, Ingrid, Joel: Tänk vad kul man kan ha. (Vin är gott i lagom mängder.)

Min växande familj:

Mamma och Pappa för att ni alltid har trott på mig.

Pim, Jecca och charmtrollet Rebecca: Pim för att du alltid har varit min storebror som hållit koll på läget, Jecca och Rebecca: Vilka Brudar!

Oscar: för att du alltid kommer att vara min lillebror hur gammal du än blir. Lycka till med dina val i livet.

Mormor: Du har alltid varit min största påhejare. Lika bra forskare som Morfar en gång var kommer jag aldrig att bli, men jag duger endå.

Min ”Nya familj”: Lisbeth och Dan, Tack för att ni låter mig låna Erik. Richard och Ulla: Ni är lika mycket mina vänner. Bättre än så kan det inte bli.

Sist men inte minst: Min ledsagare och kärlek, Erik Rönnqvist: Tack för att du alltid finns där och gör mig glad. För att du stått ut med alla helger som jag har gått iväg till labbet ”bara 5 minuter”. För att du har lärt mig att älska Fjällen. - Nu är det nya tider på gång. Snart är vi tre som ska dela på det goda i livet.

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