The abasic sites

The abasic sites (3, Scheme 8.2) resulting from the loss of alkylated bases from DNA are both cytotoxic and mutagenic. " The cyclic acetal (3) exists in equilibrium with small amounts (—1%) of the open chain aldehyde (4). The acidic nature of a-proton in the aldehyde form of the abasic lesion facilitates 3-elimination of the 3 -phosphate residue to yield a strand break. " This reaction occurs with a half-life of about 200 h under physiological conditions (pH 7.4, 37°C), but can be accelerated by heat, basic conditions, or the presence of various amines. " ... 

Figure 2a compares the time-resolved Stokes shift of the normal sequence and the abasic sequence. For ease of comparison, the data is shifted to overlap the sequences at early times. In the first nanosecond, the Stokes shifts from both sequences overlap almost perfectly. This results suggests that there is not a large scale collapse of the normal DNA structure at the abasic site. However after 1 ns, the abasic sequence has additional dynamics beyond those of the normal sequence. The fit of the abasic sequence has the same logarithmic component of the normal sequence fit, but with an additional exponential term for the fast rise in the Stokes shift after 1 ns S(t) = S0 + A0 logl0(t/t0) + 4,(l-exp(-f/r)), with an exponential time constant r of 25 ns. 

An interesting candidate for this motion is suggested by the molecular dynamics simulations of Barsky et al [11]. On a nanosecond time scale, they see a flipping of the abasic sugar from an intrahelical to an extrahelical position. This flip is correlated with a filling of the abasic site with water. Because our probe is sensitive to local polarity, it should be quite sensitive to this process. 

DNA and PNA/DNA duplexes was examined. The 32P-labeled DNA target strand was 5 -TCGCTGGcAAZAAGGbTAGGaA-3 where Z = an abasic site and three different 5 -GG-3 sites were present (GGA-C). The PNA strand s base sequence was complementary to the DNA target strand s sequence with the exception that anthraquinone (AQ) was bound to the PNA strand in place of a normal DNA base opposite the abasic site (Z) in the DNA target strand. 

Irradiation of the above PNA-DNA duplex showed preferential strand cleavage at the 5 G of the GGA and GGB sites, which was much the same result as occurred in DNA/DNA duplexes under similar conditions. Somewhat in contrast to this result, DNA strand cleavage occured equally at each G of the GGC site. The 5 -G of GGA was 6 bases away from the abasic site (ca. 20.4 A), where AQ was intercalated, yet as much damage occured there as at the 5 -G of GGB and each G of GGC. Both of these latter two sites were only two bases away from the AQ site thus distance from the site of photoexcitation was not an imporatant factor in determining strand cleavage. 

Figure 21-30. The potential energy surface along the pathway leading to the formation of the abasic site (Pabasic). The energy is in kcal/mol-1, except when otherwise indicated (Scheme 2 and Figure 2 of ref. [78]. Reprinted with permission.)...

Figure 5 Base excision repair in mammalian cells. In base excision repair, the damaged nucleotide is removed in a two-step process. A glycosylase cleaves the glycosidic bond of the damaged base, releasing it, and then AP lyase and AP endonuclease cleave the phosphodlester bonds on either side of the abasic site, which releases the deoxyribose.

An intrastrand cross-link formed by cisplatin and adjacent guanine residues causes an unusally distorted base pair (bp) step, known as the Lippard bp step. A study of the effects of neighbouring nucleotides to the cross-linked G G has shown that the 3 -nucleotide has little effect, but the 5 -nucleotide has a dramatic effect. The 5 residue always maintains an S pucker, but the canting varies, depending on the substituent. Bleomycin causes two major lesions to DNA, formation of a 4 -keto abasic site and strand cleavage to yield a 3 -phosphog-lycolate and a 5 -phosphate. As a model for the 4 -keto abasic site, an NMR structure of a 13-mer DNA duplex containing an abasic site has been reported." It was found that for both the a- and p-anomers, the abasic site was extrahelical, and that the duplex showed very little distortion to the backbone. This was discussed in terms of repair of such lesions in vivo. 

Other modifications include a formamide residue (191), a ring fragmentation product of thymine, which exists as either a cis or a trans conformer. Both isomers are rotated out of the helix, and the bases on either side of (191) occupy the space vacated by it. A pyrene abasic site base pair in DNA duplexes adopts the usual B-form duplex, with the pyrene residue within the duplex stacking on adjacent nucleobases. The abasic site folds back over the opposite strand to shelter the hydrophobic base from exposure to water.The photoresponsive azobenzene analogues (133, R- and 5-forms) have been incorporated into DNA for NMR analysis. Both isomers intercalate between neighbouring base pairs, and the 5-isomer exhibits more disturbance in its duplex structure which is refiected in lower Tms compared to the R-isomer. ... 

The abasic site occurs naturally as a result of DNA damage and repair. A number of synthetic analogues of the abasic site have been studied as has the effect of neighbouring bases in duplexes. " It was found that purines adjacent to... 

The presence of the probe in the abasic site is confirmed by hybridisation studies and by NMR. The authors have shown the existence of an apparent synergy with the anticancer agent BCNU. The incorporation of the hydroxyphenylben-zoxazole derivative (122) opposite an abasic site in a duplex is a reasonable mimic of a Watson-Crick base-pair.The analogue undergoes an excited state intramolecular proton transfer under photolysis conditions, which has been used to study tautomerisation within a DNA duplex. 

Depurinating adducts leave behind an abasic site on the DNA. Replicative DNA polymerase will insert an A opposite an abasic site so that depurination provides a straightforward route to either G — T or A —> T transversions when the daughter strand is replicated [34], As radical cation depurinating adducts result in N-glyco-sidic bond cleavage, repair of the abasic site becomes important. Apurinic/apyri-midinic endonuclease creates a strand break at the site of the lesion, and the gap is then repaired by phosphodiesterase, DNA polymerase, and DNA ligase [35], as discussed in Chapter 11. 

Figure 11.2 BER pathway. BER is initiated by DNA glycosylases that recognize lesions and hydrolyze the glycosidic bond linking the base to the sugar phosphate backbone. Monofunctional DNA glycosylases (top left) generate abasic site products that are cleaved at the 5 -phosphodiester bond by APE1. Bifunctional DNA glycosylases/AP lyases (top right) remove the abasic site and leave a...

BER often involves separate enzymes for glycosidic cleavage and endonucleolytic cleavage of the abasic site (apyrimidinic or apurinic, depending on the damage). 

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