Excision of a lyase-resistant oxidized abasic lesion from DNA

Abstract

The C2'-oxidized abasic lesion (C2-AP) is produced in DNA that is subjected to oxidative stress. The lesion disrupts replication and gives rise to mutations that are dependent upon the identity of the upstream nucleotide. Ape1 incises C2-AP, but the 5'-phosphorylated fragment is not a substrate for the lyase activity of DNA polymerase beta. Excision of the lesion is achieved by strand displacement synthesis in the presence of flap endonuclease during which C2-AP and the 3'-adjacent nucleotide are replaced. The oxidized abasic lesion is also a substrate for the bacterial UvrABC nucleotide excision repair system. These data suggest that the redundant nature of DNA repair systems provides a means for removing a lesion that resists excision by short patch base excision repair.

Figure 1

Representative plot of Ape1 (500 pM) incision of C2-AP in 5′-³²P-10.

Figure 2

Pol β (0.1 nM) and dTTP (50 μM) mediated extension of Ape1 incised C2-AP (5′-³²P-11, starting material) in the (A) absence or (B) presence of FEN1 (1 nM). Each data point is the average of 3 independent measurements ± standard deviation.

Figure 3

FEN1 (1 nM) excision of flap created by Pol β (0.1 nM) extension of Ape1 incised C2-AP (3′-³²P-11) in the presence of dTTP (50 μM) (A) Sample autoradiogram. (B) 3′-³²P-11 (starting material) and -2 nucleotide incision product as a function of time. Each data point is the average of 3 independent measurements ± standard deviation.

Figure 4

UvrABC incision of C2-AP (5′-³²P-12) as a function of time in comparison with Fl-dT (5′-³²P-13). Data are the average of 3 independent experiments ± standard deviation.

Scheme 1

Note: AP, F, C4-AP, L structures

Scheme 2