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We have used abasic residues at the 5' ends of P2 primers in order to reduce these artifacts.
P2s have two abasic residues at the 5' end to reduce primer-dimer formation (unpublished data).
Discussion Is the specificity of APE determined by binding 3' to an abasic site?
It can bypass 6-4 pyrimidine adducts and abasic sites and has a high frequency of wrong base incorporation.
It initiates base excision repair by removing damaged bases to create abasic sites that are subsequently repaired.
The abasic sugar-phosphate backbone is then cleaved by an apurinic/apyrimidinic endonuclease or lyase.
Further processing of the abasic site by AP endonucleases, polymerase I, and ligase then completes the repair.
They initiate repair of an abasic DNA site by cleaving the phosphodiester backbone 5' of the phosphodeoxyribose.
Pol θ extends mismatched primer termini and can bypass abasic sites by adding a nucleotide opposite the lesion.
The APE1 enzyme creates a nick in the phosphodiester backbone at an abasic(baseless) site through a simple acyl substitution mechanism.
Abasic and Amplifluor primers were synthesized in-house and purified by HPLC.
The uracil may be excised by uracil-DNA glycosylase (UNG) resulting in an abasic site.
This redundancy of binding to the 3' side is unique to APE, as is its strong binding to the DNA opposite the abasic site.
The AlkA protein removes a damaged base from the sugar-phosphate backbone by cleaving the glycosylic bond attaching the base to the sugar, producing an abasic site.
For example, Herschlag and co-workers have previously shown that a nucleobase could complement an abasic site within the hammerhead ribozyme, restoring cleavage activity [ 1 ] .
The 5' and 3' ends of the sense strand were capped with abasic inverted repeats, while a phosphorothioate linkage was incorporated at the 3' end of the antisense strand.
Both enzymes cleave only one DNA strand in a duplex and bacterial Xth cleaves ssDNA containing an abasic site [ 42 ] .
APE1/Ref-1 (APEX1) is a multifunctional protein possessing both DNA repair activity (on abasic and single-strand break sites) and transcriptional regulatory activity associated with oxidative stress.
Although motif 12 contains several highly conserved residues that, according to mutagenesis results (Table 1) contribute to APE1 activity, only His309 is very close to the abasic site in the DNA.
A combination of the two mutants, N226A and R177A, substantially reduced the ability of APE1 to bind to DNA containing an abasic site (Izumi et al., in preparation).
The product of xthA is an exonuclease that participates in DNA repair by recognizing and removing 5' monophosphates near abasic sites in damaged DNA (Demple et al., 1983).
The resulting abasic site is then recognised by enzymes (AP endonucleases) that break a phosphodiester bond in the DNA, permitting the repair of the resulting lesion by replacement with another cytosine.
In alkaline conditions, however, additional DNA structures are detected as DNA damage: AP sites (abasic sites missing either a pyrimidine or purine nucleotide) and sites where excision repair is taking place.
This abasic site (or AP, apurinic/apyrimidinic) may be copied by a translesion synthesis DNA polymerase such as DNA polymerase eta resulting in random incorporation of any of the four nucleotides.
Pol β, encoded by POLB gene, is required for short-patch base excision repair, a DNA repair pathway that is essential for repairing alkylated or oxidized bases as well as abasic sites.