Abstract
DNA repair is essential for the maintenance of genomic integrity, and evidence suggest that inter-individual variation in DNA repair efficiency may contribute to disease risk. However, robust assays suitable for quantitative determination of DNA repair capacity in large cohort and clinical trials are needed to evaluate these apparent associations fully. We describe here a set of microplate-based oligonucleotide assays for high-throughput, non-radioactive and quantitative determination of repair enzyme activity at individual steps and over multiple steps of the DNA base excision repair pathway. The assays are highly sensitive: using HepG2 nuclear extract, enzyme activities were quantifiable at concentrations of 0.0002 to 0.181 μg per reaction, depending on the enzyme being measured. Assay coefficients of variation are comparable with other microplate-based assays. The assay format requires no specialist equipment and has the potential to be extended for analysis of a wide range of DNA repair enzyme activities. As such, these assays hold considerable promise for gaining new mechanistic insights into how DNA repair is related to individual genetics, disease status or progression and other environmental factors and investigating whether DNA repair activities can be used a biomarker of disease risk.
Highlights
There are seven major DNA repair pathways in human cells, each with capacity to repair specific lesion types, and effective functioning of these is critical for cellular survival and health [1,2]
Note that we found that coupling of pre-hybridized and ligated complex to the wells resulted in unacceptably high background signal whereas in-well hybridization and ligation following coupling of the first oligonucleotide to the wells overcame this issue
We evaluated the effect of the nucleotide opposite the THF lesion by comparing the AP site incision activities of recombinant APE1 and HepG2 nuclear extract acting on oligonucleotide substrates generated by hybridizing and ligating Loo01Aflc, Loop01Cflc, Loop01Gflc or Loop01Tflc to AP02 immobilized in the assay plate wells
Summary
There are seven major DNA repair pathways in human cells, each with capacity to repair specific lesion types, and effective functioning of these is critical for cellular survival and health [1,2]. If repairing a base lesion, the first step in the BER pathway is performed by a DNA glycosylase that removes the damaged base. UDGs remove the damaged base and leave an AP site, which is a substrate for AP endonuclease (APE1). Following APE1 incision, BER can proceed as either short-patch BER or long-patch BER For both pathways, a polymerase acts upon the one-nucleotide gap, most commonly DNA polymerase  (POLB), adding one undamaged base in short patch BER and using its dRP lyase activity to remove the 5 dRP from the DNA backbone at the repair site [9]. More than one nucleotide is replaced via long-patch BER [10] with the resulting displaced DNA strand being excised by flap endonuclease (FEN1) [11]. The final step in both short and long patch BER is the rejoining of the DNA backbone by either DNA ligase IIIa or DNA ligase I [12,13]
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