Distinct kinetics of human DNA ligases I, IIIalpha, IIIbeta, and IV reveal direct DNA sensing ability and differential physiological functions in DNA repair Page: 4 of 29
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large number of other proteins that interact either directly with the DNA ligase polypeptide or
indirectly via their partner protein have been identified, and the functional consequences of their
interactions characterized [1]. Despite the fact that DNA ligases I and Ill are predominantly
involved in joining DNA nicks whereas DNA ligase IV completes the repair of DNA double
strand breaks in the major non-homologous end joining pathway [1,11,12], relatively few studies
have addressed the catalytic activities and substrate specificities of the human DNA ligases.
To date, most biochemical studies on human DNA ligases have utilized a radiolabeled
DNA substrate to measure DNA joining. In these assays, the conversion of the radioactively
labeled oligonucleotide or polynucleotide substrate into a labeled, higher molecular weight
product by ligation is detected after separation of the substrate and products by gel
electrophoresis. A limitation of gel-based ligation assays is that they are not suitable for carrying
out large numbers of reactions. Recently, we developed a fluorescence-based assay that we
used to screen and identify inhibitors of the human DNA ligases [13-15]. Here, we first describe
the optimization and validation of this assay and demonstrate its suitability for kinetic analyses
of DNA ligases. Subsequently, we used this technique to characterize the substrate specificity
and catalytic properties of the DNA ligases encoded by the human LIG genes.
2. Materials and Methods
2.1 Oligonucleotide substrates
The oligonucleotides listed in Table I were purchased from Integrated DNA Technologies. To
generate the nicked DNA substrate, oligonucleotide *U, containing a 5'-terminal AlexaFluor488
(AF488) dye, and D , containing a 3'-terminal Black Hole Quencher-1 (BHQ1) group, were
annealed to the complementary 40-mer oligonucleotide, T. This yielded a duplex DNA with a
single nick in the AF488+BHQ1-labeled strand, positioning the fluorophore and dark quencher
moieties 40 nucleotides apart (Fig. 1A). The double strand break (DSB) DNA substrate was
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Chen, Xi; Ballin, Jeff D.; Della-Maria, Julie; Tsai, Miaw-Sheue; White, Elizabeth J.; Tomkinson, Alan E. et al. Distinct kinetics of human DNA ligases I, IIIalpha, IIIbeta, and IV reveal direct DNA sensing ability and differential physiological functions in DNA repair, article, May 11, 2009; Berkeley, California. (https://digital.library.unt.edu/ark:/67531/metadc926316/m1/4/: accessed April 24, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; crediting UNT Libraries Government Documents Department.