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. 2016:2016:3125989.
doi: 10.1155/2016/3125989. Epub 2015 Nov 16.

Efficiency of Base Excision Repair of Oxidative DNA Damage and Its Impact on the Risk of Colorectal Cancer in the Polish Population

J Kabzinski  1 , B Mucha  1 , M Cuchra  1 , L Markiewicz  1 , K Przybylowska  1 , A Dziki  2 , L Dziki  2 , I Majsterek  1
Affiliations

Efficiency of Base Excision Repair of Oxidative DNA Damage and Its Impact on the Risk of Colorectal Cancer in the Polish Population

J Kabzinski et al. Oxid Med Cell Longev. 2016.

Abstract

DNA oxidative lesions are widely considered as a potential risk factor for colorectal cancer development. The aim of this work was to determine the role of the efficiency of base excision repair, both in lymphocytes and in epithelial tissue, in patients with CRC and healthy subjects. SNPs were identified within genes responsible for steps following glycosylase action in BER, and patients and healthy subjects were genotyped. A radioisotopic BER assay was used for assessing repair efficiency and TaqMan for genotyping. Decreased BER activity was observed in lymphocyte extract from CRC patients and in cancer tissue extract, compared to healthy subjects. In addition, polymorphisms of EXO1, LIG3, and PolB may modulate the risk of colorectal cancer by decreasing (PolB) or increasing (LIG3 and EXO1) the chance of malignant transformation.

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Figures

Figure 1
Figure 1
(I) A uracil-containing oligonucleotide was subjected to action of polynucleotide kinase to attach a radioactive phosphate group from [γ-32P] ATP. It was hybridized with a second oligonucleotide whose sequence was adjusted to obtain sticky ends referring to XhoI and XbaI digestion site. (II) The short DNA fragment prepared in stage I was cloned into a pBluescriptII plasmid. (III) Uracil-DNA glycosylase was utilized to remove uracil and, as consequence, create a single gap in DNA to act as a synthetic lesion. (IV) A plasmid with single AP site constituted a substrate for the protein extract in 90-minute repair incubation. (V) Two SacI recognition sites of the pBluescriptII plasmid were used to excise 450 pb-long fragment covering the lesion site and radioactive label for analysis on 8% urea/acrylamide gel. (VI) Interpretation of outcomes was based on detection of two bands. The full-length 450 pb fragment reflects restored DNA fraction, whereas presence of short 180 pb fraction indicates the amount of unrepaired DNA. ∗U: uracil; ∗∗AP: apurinic/apyrimidinic.
Figure 2
Figure 2
A comparison of BER activity in the lymphocytes and tissue of CRC patients and healthy controls. Each electropherogram shows two fractions of DNA: 450 pb repaired and 185 pb unrepaired. Lanes 1-2 indicate lymphocyte BER efficiency while lanes 3-4 refer to BER in tissue. Samples are presented in the following order: K: positive control; DNA substrate did not contain uracil and so reflects 100% of repair; 1: healthy control; 2: colorectal cancer; 3: unchanged colon tissue; 4: colorectal cancer.
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