Dual-incision

The mechanism of eukaryotic excinucleases is quite similar to that of the bacterial enzyme, although 16 polypeptides with no similarity to the E. coli excinuclease subunits are required for the dual incision. As described in Chapter 26, some of the nucleotide-excision repair and base-excision repair in eukaryotes is closely tied to transcription. Genetic deficiencies in nucleotide-excision repair in humans give rise to a variety of serious diseases (Box 25-1). 

The dual incisions results in the release of an oligonucleotide containing the damaged base that is on average about 27 nucleotides in length. The gap that is formed is filled in by either DNA polymerase 8 or DNA polymerase e in reactions dependent upon PCNA. DNA ligase I seals the final nick. 

Figure 6 Nucleotide excision repair in (A) E. coli and (B) humans. There are five basic steps of nucieotide excision repair (1) damage recognition, (2) dual incisions, (3) release of the excised oligomer, (4) repair synthesis to fill in the gap, and (5) ligation.

Evans E,Moggs JG, Hwang JR,EglyJM,Wood RD (1997) Mechanism of open complex and dual incision formation by human nucleotide excision repair factors. EMBO J 16 6559-73... 

An additional level of coordination is likely achieved through a temporal coordination of the two dual-incision reactions. Studies using catalytically inactive versions of ERCC1 and XPG have shown that 5 -indsion by XPF requires the presence, but not catalytic activity of XPG [85, 86], while efficient 3 -indsion by XPG only occurs following 5 -indsion by XPF [71, 93], Furthermore, repair synthesis can be initiated in the presence of catalytically inactive XPG, providing another possible mechanism ensuring that exposure of persistent single-stranded DNA intermediates is actively prevented [93],... 

Wakasugi, M., Reardon, J.T., and Sancar, A. (1997) The non-catalytic function of XPG protein during dual incision in human nucleotide excision repair./. Biol. Chem., 272,16030-16034. 

Analysis of the roles of the subunits involved in dual incisions by using anti-XPG and anti-ERCCl antibodies. 

J. H., Gourdin, A.M., Wijgers, N., Dunand-Sauthier, I., Giglia-Mari, G., Clarkson, S.G., Vermeulen, W., and Scharer, O.D. (2009) Coordination of dual incision and repair synthesis in human nucleotide excision repair. EMBOJ., 28, 1111-1120. 

Figure 12.1 Outline of NER assay in human cell extracts. The hallmark of a successful mammalian NER dual-incision activity is the excision of internally 32P-labeled 24- to 32-nucleotide fragments containing the...

The NER pathway is also present in prokaryotic organisms, such as Escherichia coli, as well as in eukaryotes from yeast to mammals. Many of the basic steps of NER are evolutionarily conserved, including damage recognition and dual incisions to excise the lesion, helicase activity to displace the excised oligonucleotide and repair factors, and synthesis and ligation enzymes to seal the nick [19, 20], Nevertheless, the biochemical features in prokaryotes and eukaryotes are distinct. 

DNA duplex. Naegeli et al. have previously advanced a bipartite model of NER substrate discrimination that is initiated by the detection of disrupted Watson-Crick base-pairing followed by a lesion-sensing step that verifies the presence of a chemically altered nucleotide [29, 33]. The nature of the critically important verification step that leads to the dual incision is still not well understood [24]. The bipartite model is consistent with previous observatisons of Sugasawa et al. who found that XPC/HR23B binds to DNA that contains bubbles of several mismatched DNA bases in the absence of lesions or chemically modified nucleotides, but incisions occur only when a chemically modified base is also present [13, 35]. 

Figure 12.5 (a) Autoradiographs of dual excision products NER of (+)-cis-l, (+)-trans-l, and (-)-trans-/ 135mer duplexes in the 5 -.., CCATCG CTACC... sequence context (CC C-I in Figure 12.9) hybridized with a fully complementary strand with C opposite G. The duplexes were internally 32P 5 -end-labeled at the first C shown here at the sixth phosphodiester bond on the 5 -side of the B[o]P-N2-dG lesions (G ). The dual-incision products were obtained after incubation of the 135mer duplexes containing different stereoisomeric G lesions with nuclear extracts from human HeLa cells for 30 min at... 

In order to compare the relative NER efficiencies of our PAH diol epoxide-DNA adducts we prepared site-specifically modified oligonucleotides containing either CPD or 6-4 photodimers using the same sequence and methods as described in the literature [110], Typical results obtained in our laboratory are displayed in Figure 12.6(a). There are fewer NER dual-incision bands in the case of the CPD dimer than in the case of the 6-4 photoproduct and the latter are clearly more intense than the former. Densitometry analysis shows that the time course of dual incisions is linear at least up to 40 min and that the DNA strand bearing the 6-4 adduct is around 17 times faster than the strand with the CPD dimer (Figure 12.6c). The repair rate of the (+)-cis-anti-B[a] P-N2-dG adduct and the 6-4 photoproduct coincide with one another (Figure 12.6c), as described in more detail elsewhere [78],... 

The sequence-dependent trend is conserved in the prokaryotic UvrABC system, in which the NER dual-incision efficiency is around 2.4-fold higher for the same 10S (+)-trans-anti-B[a]P-N2-dG adduct in the TG T case than the CG C-II case [124]. Similarly, the G6 G7 duplex is incised more efficiently by a factor of around... 

Y., Rodriguez, F., Kolbanovskii, A., Liu, Y., Zhang, L., Amin, S., Patel, D., Broyde, S., et al. (2009) The sequence dependence of human nucleotide excision repair efficiencies of benzoja] pyrene-derived DNA lesions insights into the structural factors that favor dual incisions. J. Mol. Biol., 386, 1193-1203. 

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