DCTP insertion

These observations provide a reasonable rationale for why Pol IV preferentially does cellular dCTP insertion in cells, since a reasonable adduct-dG dCTP structure emerges with near reaction-ready distances between primer-3 -0 and Pa-dCTP. 

Figure 10.3. Mass array, (a) Primer binding (b) primer extension enzyme, ddATP and dCTP/ dGTP/dTTP addition (c) primer terminates (d) primer extension products ready for MALDI-MS (e) MS spectrum of primer extension products. Each addition of a nucleotide to the primer extension product increases the mass by 289 to 329 Da, depending on the nucleotide added. The mass difference is easily resolved by MALDI-TOF, which has the ability to detect differences as small as 3 Da. Printed by kind permission of Sequenom. (See color insert.)...

Our LIC scheme uses dCTP in the vector sample and dGTP in the insert sample to create the single-stranded overhangs. This can vary, depending on the LIC sequence that is being used. 

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). 

Translesion synthesis with DNA Pol of the A-acetyl-2-aminofluorene adduct of guanosine (88) is inefficient with templates containing (88). In the presence of the Revl protein, translesion synthesis occurs and dCTP is the major nucleotide incorporated opposite it, and studies with a mutant DNA Pol I gave similar results. Benzo[a]pyrene is a potent environmental carcinogen, which when metabolised leads to u t -benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide anti-BPDE). With dG, the major lesion is (+)-tra w-a h-B[a]P-A -dG, (89), and is usually repaired by the nucleotide excision repair (NER) pathway. The translesion synthesis past (89) has been examined with a number of polymerases. With human RNA Pol II, (89) is a block to synthesis, whilst DNA Pol k preferentially incorporated the correct nucleotide. In yeast cells, Pol induced a large number of mutations involving Pol p, whilst Pol p alone contributed to 1-3 deletions or insertions. The NER of (89) with UvrB proteins was also studied. ... 

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... 

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]. 

The extreme level of BF inhibition is in contrast to steady-state analysis of human Pol 8. The degree of inhibition by 06-MeG observed for both the replicative Pol 8 (with the sliding clamp) and three recombinant Y-family polymerases (T, I, and k) was found to be relatively modest (10- to 100-fold) [149], With one exception, all of the human enzymes tested in the aforementioned study incorporated dCTP and dTTP equally well opposite 06-MeG. The exception to this pattern was human Pol t, which performed insertion of dTTP opposite 06-MeG 10 times better than dCTP opposite the lesion and with greater efficiency than dCTP opposite G (around 2-fold). It is entirely possible that inclusion of accessory proteins and/or post-translational modifications to the Y-family members could influence the catalytic efficiency of (/ -MeG bypass. In vitro studies with all polymerases studied to date verify the mutagenic potential of the 06-MeG adduct. 

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