Molecular Cloning

All molecular cloning was performed in a separate room, surfaces and material disinfected prior to use with 70 % EtOH. Glassware, buffers and solutions were sterilized (3 h at 180°C) or autoclaved, respectively. All work was done close to an open flame and all toothpicks, metal- and glassware was flame sterilized directly before usage.

3.2.2.2 Preparing Chemically Competent DH5α Cells, of LB Agar and Agar Plates

Chemo competent DH5α cells were generated with the Mix & Go E. coli transformation kit according to the manufacturer’s instructions.

LB-Miller medium (0.5 % Bacto Yeast Extract; 1 % Bacto Tryptone; 10 g/L NaCl) and LB agar (0.5 % Bacto Yeast Extract; 1 % Bacto Tryptone; 1.5 % Bacto Agar; 10 g/L NaCl) were prepared, autoclaved and stored at 4°C in the dark. To cast LB agar plates, LB agar was heated in a microwave until being completely liquid. The LB agar was cooled to about 50°C before adding kanamycin (100 µg/ml) or ampicillin (40 µg/ml) and casting into 100 mm petri dishes.

3.2.2.3 Polymerase Chain Reaction (PCR)

For primer design it was taken care to have a GC content close to 40-60 %, a 3’ GC clamp, single base runs were avoided as well as secondary structures and intra-primer homologies. Standard primers were designed to length of 18-30 bases, for site-directed mutagenesis and the introduction of new base stretches primer length was extended up to 45 bases. A 5’ extension of at least 6 bases was added to an introduced restriction cutting site. Primers for site-directed mutagenesis carried the base mismatches close to their middle region. Subsequent rounds of site-directed mutagenesis were performed when multiple bases (>4) had to be exchanged. Forward and reverse primer were designed in a manner that their Tm did not differ more than 5°C. Tm was calculated according to the OligoAnalyzer tool. A Kod Hot Start DNA polymerase was used according to the manufacturer’s instructions.

3.2.2.4 Transformation of Chemically Competent DH5α

Chemocompetent DH5α E.coli cells were thawed on ice and mixed by gently pipetting up and down. 5 ng pure plasmid DNA or 5 to 10 µl ligation or PCR reaction mix were added to 50 µl DH5α in 1.5 ml reaction tubes. After careful mixing, the tubes were incubated for 30 min on ice. Thereafter, a heatshock for 20 s at 42°C was performed and the cells were again incubated on ice for 2 min. 1 ml of prewarmed LB medium, without antibiotic, was added. E. coli cells were kept for 1 h at 37°C on a thermal shaker (500 rpm) to allow the expression of the antibiotic resistance selection marker encoded on the transformed plasmid. When pure plasmid DNA was used for the transformation, either 10 µl and 50 µl bacterial suspension were plated on LB agar plates with the appropriate antibiotic or used to inoculate a ~4 ml culture (+appropriate antibiotic) for a subsequent mini preparation or as a starter culture. When

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PCR or ligation mixtures were used for transformation, 100 µl was plated on LB agar plates. The remaining solution was centrifuged down (1 min, 12,000 rpm), the bacterial pellet resuspended in 100 µl LB medium and as well streaked on LB agar. The plates were incubated at 37°C and on the next day, single colonies were picked with a toothpick. 4 ml cultures (+appropriate antibiotic) were inoculated in test tubes and incubated at 200 rpm overnight.

3.2.2.5 Plasmid DNA Purification

Mini preparation of plasmids from 4 ml E. coli overnight cultures was performed with the ZR Plasmid Miniprep Kit-Classic according to the manufacturer’s instructions.

For midi, maxi and giga plasmid preparations a 4 ml starter culture of one single bacterial colony was incubated for at least 8 hours (200 rpm, 37°C). The starter culture was diluted 1:500 in LB medium with the appropriate antibiotic and incubated at 37°C with vigorous shaking (200 rpm) to an OD600 of about 1.5.

Midi preparation of plasmids from 25 ml E. coli overnight cultures was performed with the QIAGEN Plasmid Midi Kit according to the manufacturer’s instructions. Maxi preparation of plasmids from 100 ml E. coli overnight cultures was performed with the PhoenIX Filter Maxiprep Kit or the QIAGEN Plasmid Plus Maxi Kit according to the manufacturer’s instructions. Giga preparation of plasmids from 500 ml E. coli overnight cultures was performed with the QIAfilter Plasmid Mega and Giga Kit according to the manufacturer’s instructions.

Plasmid concentration and purity (A260/A280) was defined with a ND-1000 nanodrop. Plasmid and plasmid-insert identity was checked by either analytic restriction digest or by sequencing with appropriate primers (GATC, LIGHTRUN sequencing).

3.2.2.6 Analytical an Preparative Plasmid DNA Digestion

To verify the identity of a plasmid, ligation or PCR product an analytic DNA digestion was performed.

0.5-1 µg DNA, depending on the fragment lengths, was digested with the appropriate amount of restriction enzyme according to the manufacturer’s instructions. Preparative DNA digestion was done as described above, but with DNA amounts up to 100 µg. After digestion, DNA was run on an agarose gel, to check the band pattern for analytic, and to gel purify for preparative digest.

3.2.2.7 Agarose Gel Electrophoresis

To quality control PCRs, plasmid amplifications and restriction digestions DNA was size separated with agarose gel electrophoresis. Therefore, DNA was run in 1-4 % agarose, depending on the DNA length at 120 V as long as needed. Subsequently, the gel was incubated for 30 min in a 0.5 µg/ml ethidium bromide bath and washed afterwards for 30 min in DI water. DNA was then visualized by fluorescence detection of intercalated ethidium bromide in a UV chamber (Ex.: 312 nm).

3.2.2.8 DNA Gel Extraction

DNA purification from agarose gels was used after restriction digests of plasmids to isolate and purify DNA. Therefore, it was possible to separate for example cut out inserts from its plasmid backbones and subsequently re-ligate them into another vector.

For DNA gel purification, the restriction digest was run in a low percent agarose gel (<1.5%) at 120 V.

The gel was stained by incubation in a 0.5 µg/ml ethidium bromide bath (30 min) and washed for 30 min in DI water. Intercalated ethidium bromide was visualized on an UV fluorescence table (Ex.: 300).

The target band was identified based on its size and cut out. DNA gel extraction was performed with the QIAquick Gel Extraction Kit according to the manufacturer’s instructions.

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Ligation of an insert into a vector backbone was performed in 15 µl reaction approaches. Here, one part vector was mixed with five parts insert and incubated on a thermoxixer for 5 min at 55°C and subsequently 1 min on ice. Thereafter, T4 DNA ligase and ligation buffer was added according to the manufacturer’s instructions. Ligation proceeded overnight at 4°C. The next day, the DNA ligase was inactivated by incubation of the mixture at 65°C for 15 min.

3.2.2.10 Site-Directed Mutagenesis (SDM)

Based on the results of the in-silico PBM analysis and the subsequent in-vitro peptide PAR-binding studies a site-directed mutagenesis in the PBM of XPA was conducted. The aim was to generate constructs, coding for PAR-binding-deficient XPA-eGFP for further analysis of the PAR-dependent spatio-temporal regulation of XPA in the cell.

The pEGFP-N1::XPA-eGFP plasmid (10 ng) was used as template for the site-directed mutagenesis. It was aimed at the exchange of three basic amino acids in the PBM of XPA to alanine (XPA-PBM_mut2).

Therefore two subsequent rounds of site-directed mutagenesis were performed (see Figure 4.27). In a first step R227 and R231 were exchanged (Primer: SDM 12f and SDM 12r), in a second step R228 (Primer: SDM 13f and SDM 13r).

Additionally, a second mutant version of the XPA-eGFP was generated (XPA-PBM_mut1) as described above (first step: K217 and K221; second step: K218 and K222).

Table 3.1: Thermocycling parameters site-directed mutagenesis.

Step Temperature Time

1. Initial denaturation 95°C 120 s

2. Denaturation 95°C 20 s

3. Primer annealing 68°C 20 s

4. Elongation 70°C 170 s

5. Final elongation 70°C 10 min

6. Storage 8°C ∞

Steps 2-4 were repeated 22 times

After the site-directed mutagenesis a Dpn1 digest was conducted. By doing so, the methylated template DNA was removed and only SDM-PCR originated DNA remained. Before E. coli transformation (3.2.2.4), the reaction mix was heat- inactivated (20 min at 80°C). The transformed E. coli were plated, single colonies were picked and 4 ml bacterial cultures were grown. Mini preparations were performed and the plasmid sequence was analysed by sequencing. SDM positive plasmids were used as a template for the second round of SDM-PCR.

3.2.2.11 Cryoconservation of DH5α Cells

For long term storage of already transformed E. coli cultures, glycerol stocks were prepared. A 4 ml bacterial culture was grown overnight in the appropriate antibiotics. The cell suspension was centrifuged for 10 min (3,000 rpm; 4°C) and the supernatant was discarded. The cell pellet was resuspended in LB medium supplemented with glycerol to a final concentration of 20 % and stored at -80°C.

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In-silico search for PBMs in peptides was performed with a basal pattern of alternating basic and hydrophobic amino acids (BxHHBBHHB) using the NPS@ algorithm PATTINPROT (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?pa ge=npsa_pattinprot.html). Sequences used to align XPA’s PBM of different species were obtained from www.ensembl.org. Peptide sequence alignments were performed with Geneious software.

3.2.3 Recombinant Protein Overexpression and Purification