Clustered DNA Lesions Containing 5-Formyluracil and AP Site: Repair via the BER System

<div><p>Lesions in the DNA arise under ionizing irradiation conditions or various chemical oxidants as a single damage or as part of a multiply damaged site within 1–2 helical turns (clustered lesion). Here, we explored the repair opportunity of the apurinic/apyrimidinic site (AP site) composed of the clustered lesion with <u>5-fo</u>rmyl<u>u</u>racil (5-foU) by the base excision repair (BER) proteins. We found, that if the AP site is shifted relative to the 5-foU of the opposite strand, it could be repaired primarily via the short-patch BER pathway. In this case, the cleavage efficiency of the AP site-containing DNA strand catalyzed by human <u>ap</u>urinic/<u>ap</u>yrimidinic <u>e</u>ndonuclease <u>1</u> (hAPE1) decreased under AP site excursion to the 3'-side relative to the lesion in the other DNA strand. DNA synthesis catalyzed by DNA polymerase lambda was more accurate in comparison to the one catalyzed by DNA polymerase beta. If the AP site was located exactly opposite 5-foU it was expected to switch the repair to the long-patch BER pathway. In this situation, human processivity factor hPCNA stimulates the process.</p></div

Specificity of dNMP incorporation catalyzed by DNA polymerases beta and lambda in the presence of Mg²⁺ (A) or Mn²⁺ (B) ions using DNA1 (lanes 1-10), DNA2 (lanes 11–20) and DNA3 (lanes 21–30).

<p>Lane C, position of the 5'–(³²P)labeled 55 and 18 nt-long DNA; lanes 1, 11 and 21, initial DNA substrate after UDG treatment; lanes 2, 12 and 22, initial DNA substrate after sequential treatment by UDG and hAPE1; 18, 19 and 20 nt, lengths of the reaction products. The autoradiographs present the results from one of three independent experiments.</p

Efficiency (k_cat/K_m, μM⁻¹⋅sec⁻¹) of dNMPs incorporation by DNA polymerases beta and lambda via the long-patch BER pathway.

<p>Note: the results are presented as the average value of three independent experiments. Standard error was estimated as 10%.</p

BER pathways for repair of AP site introduced into the cluster damage with 5-foU in the opposite strand.

<p><b><i><u>U^f</u></i></b>, 5-foU, <b><i><u>O</u></i></b>, AP site.</p

Strand-displacement activity of DNA polymerases beta (A, B) and lambda (C, D) in the presence of Mg²⁺ (A, C) or Mn²⁺ (B, D) ions using DNA1 (lanes 1–6), DNA2 (lanes 7–12), DNA3 (lanes 13–18) and DNA4 (lanes 19–24).

<p>Lane C, position of the 5'–(³²P)labeled 55 and 18 nt-long DNA; lanes 1, 7, 13 and 19 (upper panel), initial DNA substrate after UDG treatment; lanes 2, 8, 14 and 20 (upper panel), and 1, 7, 13 and 19 (bottom panel), initial DNA substrate after sequential treatment by UDG and hAPE1; 18, 19 and 20 nt, lengths of the reaction products. The final concentrations of dNTPs are indicated below the gels. The autoradiographs present the results from one of three independent experiments.</p

Efficiency (k_cat/K_m, μM⁻¹⋅sec⁻¹) of dNMP incorporation by DNA polymerases beta and lambda via the short-patch BER pathway.

<p>Note: the results are presented as the average value of three independent experiments. Standard error was estimated as 10%.</p

Efficiency (kcat/Km, 1/nM⋅sec) of the DNA endonuclease cleavage of the AP-DNA strand by hAPE1.

<p>Empty and shaded columns designate the values obtained in the presence of Mg²⁺ and Mn²⁺ ions, respectively. The results are presented as the average value of four independent experiments. Standard error was estimated as 10%.</p

Influence of hXRCC1 on the endonuclease activity of hAPE1 (A), and on the incorporation of dNMP catalyzed by DNA polymerases beta (B) and lambda (C).

<p>The data are presented in logarithmic scale. Solid lines designate the yield of the reaction products obtained in the presence of Mg²⁺ ions; dotted lines designate the yield of the reaction products obtained in the presence of Mn²⁺ ions. The yield of the reaction products is the ratio of the reaction products over the initial amount of substrate expressed as a percentage. dGTP was used for the reactions with DNA1 and DNA3, and dATP – for the reactions with DNA2 and DNA4. The results are presented as the average value of four independent experiments. Standard error was estimated as 10%.</p

Influence of hPCNA on long-patch BER activity of DNA polymerases beta and lambda using DNA1 (A), DNA2 (B), DNA3 (C) and DNA 4 (D).

<p>Lane C, position of the 5'-(³²P)labeled 55 and 18 nt-long DNA; lane 1, initial DNA substrate after UDG treatment; lanes 2 and 6, initial DNA substrate after sequential treatment by UDG and hAPE1; 18, 19 and 20 nt, lengths of the reaction products. The final concentrations of hPCNA are indicated below the gels. The autoradiographs present the results from one of three independent experiments.</p