DATP insertion

Dpo4 (from S. solfataricus) show unaltered or enhanced efficiency when bypassing 8-oxo-G [90, 93], Both of these enzymes bypass 8-oxo-G in a highly accurate manner, with a 20-fold preference for dCTP over dATP. The eukaryotic DinB homolog Pol K is very error-prone when catalyzing nucleotide incorporation opposite 8-oxo-G, showing much greater preference for dATP insertion [58]. 

Although Pol V replicates undamaged templates with relatively low fidelity (10 3 to 10-4) [76], one striking quality is Pol V s ability to accurately bypass UV photoproducts (e.g., inserting dATP opposite thymine-thymine (TT) cyclobutane pyrimidine dimers (CPDs) [76]). Analysis of insertion tendencies opposite a variety of adducts/lesions led to the observation that Pol V seems to have two insertion modes (i) correct dNTP insertion and (ii) default dATP insertion [37]. UV light is a frequently encountered form of DNA damage for which a translesion synthesis DNA polymerase might be important and since TT CPDs are the major UV lesion... 

DNA synthesis is catalysed by DNA polymerases and requires the precursor dNTPs (dATP, dGTP, dCTP and dTTP, each of these existing as Mg2+ complexes), a template (i.e. the dsDNA being copied) and a primer (an initial deoxyribose 3 -OH to enable the reaction to insert the first new nucleotide). The reaction proceeds in a 5 to 3 direction, that is, at the end of the synthesis there is a vacant deoxyribose 3 -OH. The fidelity of the replication process is based on the incoming nucleotides base pairing with the correct base on the antiparallel template. DNA synthesis is semi-conservative (i.e. the newly synthesized strand partners its antiparallel complementary strand) and is bidirectional (because both original strands are replicated). 

Figure 10.16 Steady-state kinetic efficiencies for single-nucleotide insertion by HIV-1 reverse transcriptase to duplex RNA templates [5 -r(ACU GXU CUC CCU AUA GUG AGU CGU AUU A),-55 -d(T AAT ACG ACT CAC TAT AGG GAG A)] with dATP.

Mutagenesis studies showed that this type of mutation induced a -1 frameshift. A further report describes the incorporation of an (adjacent) cis-syn cyclobutane dimer site specifically into a nucleosome The templating properties of the thymine photoproducts, including the Dewar photoproduct (176), have been examined using the pyrene C-nucleoside triphosphate. The pyrene nucleotide was inserted in preference to dATP opposite the 3 -T of the photoproducts in all cases except for a trans,syn photoproduct, whereas dATP was preferentially incorporated opposite the 5 -T in all cases. This suggests that the incorporation opposite the 3 -end of the photoproduct takes place via a transient abasic site-like intermediate. 

Polymerase bypass of the mutagenic lesion 8-oxo-G has been studied in considerable detail. All DNA polymerases studied to date insert either dCTP or dATP opposite 8-oxo-G [82-90], Relatively modest decreases in catalytic efficiency have been observed for some polymerases (e.g., around 30-fold decrease for E. coli Pols I and II (exo ), around 10-fold for calf thymus Pol 8), whereas the catalytic efficiency of the model B-family polymerase from bacteriophage T7 (Pol T7) was inhibited around 270-fold as judged by pre-steady-state measurements [83, 85, 86, 91, 92], Only the Y-family polymerases Pol T (from Saccharomyces cerevisiae) and... 

The 6-4 lesions generated by exposure to UV-B radiation strongly perturb duplex DNA [199], Both yeast and human Pol q can insert dGTP opposite the 3 -T in a 6-4 lesion, albeit 50- to 100-fold less efficiently than with an undamaged template [200]. The low-fidelity B-family member Pol C, can apparently insert dATP opposite the 5 -T of a 6-4 lesion with very little reduction in catalytic efficiency [200], In contrast to Pol q, Pol t prefers to accurately insert dATP opposite the 3 -T of a 6-4 lesion [201], Therefore, accurate bypass of 6-4 lesions may be achieved by the combined action of Pols t and C,. 

Pol V is involved in other mutagenesis pathways for example, it inserts dATP opposite +BP in the G — T mutational pathway ([79], discussed below). In fact, the preferential mutagenic insertion of dATP opposite a variety of DNA lesions in E. coli has been called the A-rule ([60, 80] and references therein) and it seems likely that this is attributable to Pol V s tendency to insert dATP [37]. Based on lesion-bypass specificity, E. coli Pol V appears to be the functional ortholog of human Pol q [37], which is almost certainly responsible for correct bypass of UV lesions in human cells and minimizes UV light mutagenesis that leads to skin cancer [81-86]. 

When replicative DNA polymerase encounters a thymine dimer, it cannot replicate past the site. Deoxyadenylate is incorporated opposite the first thymine base in the template, but the double helix distortion induced by the thymine dimer causes the structure to be recognized as a mismatch, and the polymerase "idles" at the damage site, converting dATP to dAMP by a continual process of insertion and exonucleolytic cleavage (due to proofreading). 

DNA to be labeled, digested to lengths of 200-400 base pairs (bp) with DNase I typically for a 2.5-3.0 kbp insert template, 1/350 unit of DNase I is used in nick translation buffer (see below) for 5 min at 37°. The time required may vary for different length templates Unlabeled dATP, dGTP, dTTP (200 pM adjusted to pH 7.0)... 

Figure 4 schematically describes the synthesis of a probe. A universal sequencing primer (e.g., 1211, BRL) is annealed to its complementary sequence in the vicinity of the M13 polylinker. Klenow polymerase is then used to elongate the primer and synthesize the complementary strand of the probe insert which will be highly labeled if [a pj dATP of high specific activity is Included in the reaction. Provided that polymerase, primer and cold nucleotides are in excess, primer elongation will start... 

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