**2.1 Comet assay**

Alkaline version of comet assay (Singh et al., 1988) with small modifications was used. Detailed procedure is described elsewhere (Milić, 2010; Milić et al., 2010). The slides were marked and stored at +4°C till the beginning of the irradiation. Control samples were put into cold lysis solution immediately after preparation and left there for 24 hours at +4°C (Milić, 2010). Irradiated blood gel samples were incubated at 37°C in serum free RPMI 1640 medium. Zero samples were immediately immersed into lysis solution. DNA repair kinetics was measured at 0, 15, 30, 60 and 120 minutes after exposure, and additional 24 hours for samples irradiated with the dose of 4 Gy. Specific measure points were based on the results of other researches (Singh et al., 1988; Price, 1993; Tice, 1995). The 2 Gy dose was a standard daily dose in radiotherapy, while the 4 Gy dose was chosen as a challenging dose for the exposed group. After the repair, slides were vertically placed into cold lysis solution at 4°C, overnight. Protein denaturation and DNA unwinding were done at 4°C in denaturation buffer (1 mM Na2EDTA and 300 mM NaOH) (pH 13.0) for 20 minutes. Horizontal electrophoresis in fresh cold denaturation buffer was done at 300 mA and 25 V for 20 minutes. The slides were washed in neutralisation buffer (0.4 M Tris-HCl, pH 7.5) three times. Slides were stained with 50 µL of ethidium bromide solution (20 μgmL-1, Sigma) (per slide), covered with cover slip and kept in container, in the dark conditions at 4°C.

The procedure was done under dimmed light, in order to avoid additional DNA damage caused by the exposure to the normal light. Each slide was examined using a 250x magnification fluorescence microscope (Zeiss, Germany) equipped with an excitation filter of 515-560 nm and a barrier filter of 590 nm. A total of 200 comets per sample and per interval were scored (100 from each of two replicate slides). Comets were randomly captured at a constant depth of the gel, avoiding the edges of the gel, occasional dead cells and superimposed comets. Using a black and white camera, the microscope image was transferred to a computer-based image analysis system (Comet Assay II, Perceptive Instruments Ltd., U.K.). To avoid the variability, one well-trained scorer scored all comets. Three parameters of DNA damage were analysed: tail length (TL, presented in micrometres), tail DNA (TI, %) and tail moment (TM).

#### **2.2 DNA isolation**

Genomic DNA was isolated from peripheral lymphocytes according to modified protocol by Daly et al. (1996) or according to protocol for genomic DNA lymphocyte isolation from QUIAGEN (mini KIT). DNA was purified with two times centrifugation at 4°C, 500 μL of 70 percent ethanol added every time. The pellet was dried overnight at room temperature and diluted in 100 μL of TE buffer (10mM Tris–HCl, pH 7.4; 1 mM EDTA, pH 8.0). Purity and concentration of DNA was specified by spectrophotometric method (NanoDrop ND- 1000 spectrophotometer, NanoDrop Technologies, Thermo Scientific, Wilmington, USA). Samples were diluted till concentration of 10 ng μL-1 and kept at -20 °C till amplification. Specific polymorphisms were determined: in BER- (base excision repair) APE1- (apurinic/apirimidinic endonuclease, Asp148Glu), hOGG1 (human 8-oxoguanine DNA glycosylase, Ser326Cys), XRCC1 (X-ray repair cross-complementing protein-group 1, Arg194Trp); in NER- (nucleotide excision repair) XPD (Xeroderma pigmentosum-group D, Lys751Gln; DSBR- (double-strand-break repair) XRCC3 (X-ray repair cross-complementing protein-group 3, Thr241Met), PARP1 (poly (ADP-ribose) polymerase 1, Val762Ala); in DRR- (direct reversal repair) MGMT (O6-methylguanine-DNA methyltransferase, Leu84Phe) Genotyping was performed by either Real Time PCR (polymerase chain reaction) with Taqman assay, or after electrophoresis and fluorescence visualisation, DNA samples were cut with restriction enzymes.
