L cell DNA ligase joins RNA to DNA on a DNA template

Abstract

L cell DNA ligase catalyzes a covalent linkage between 5'-hosphoryl oligodeoxyribonucleotides and 3'-hydroxyl oligoribonucleotides on a complementary polydeoxyribonucleotide template. This reaction occurs to a substantially lesser extent than does the sealing of DNA to DNA. The joining of [5'32P]d(pA)12-18 to (Ap)11A on poly[d(T)] or of [5'-32P]d(pG)12-18 to 5'-hydroxyl, 3'-hydroxyl oligo(I) ON POLY[D(C)] was demonstrated by the formation of alkaline phosphatase resistant radioactivity. The 32P of the hybrid reaction products became sensitive to the action of alkaline phosphatase after treatment with alkali. Furthermore, hydrolysis of the products of the linkage of [5'-32P]d(pG)12-18 to 5'-hydroxyl, 3'-hydroxyl oligo(I) on poly[d(C)] with micrococcal nuclease and spleen phosphodiesterase resulted in the formation of [3'-32P]IMP. Attempts to seal [5'-32p[-(pA)12 to d(Ap)11-17A on poly[d(T) or [5'-32P]oligo(pI) to d(Gp)11-17G on poly[d(C)] were unsuccessful.

Last Stop on the Road to Repair: Structure of E. coli DNA Ligase Bound to Nicked DNA-Adenylate

“…The A-like segment includes two base pairs flanking the nick on the 3 0 side and one base pair on the 5 0 side. The adoption of an RNA-like conformation surrounding the nick in the LigA-DNA complex echoes previous findings for the human Lig1-DNA cocrystal (Pascal et al, 2004) and accounts for the ability of DNA ligases generally to seal duplexes in which the broken 3 0 -OH strand is composed of RNA (Nath and Hurwitz, 1974;Bedows et al, 1977;Sekiguchi and Shuman, 1997b;Sriskanda and Shuman, 1998b;Pascal et al, 2004).…”

RNA primers in SV40 DNA replication: identification of transient RNA-DNA covalent linkages in replicating DNA

“…Note: The [a-32P]dATP data were corrected for a secondary source of label transfer (see text) which caused the background to increase with apparent first-order kinetics from 0.02% to 0.09% during the chase. (Kaufmann and Littauer, 1969), terminal transferase (Roychoudhury and Kossel, 1971), RNA polymerase (Hurwitz et al, 1972), and mammalian DNA ligase (Bedows et al, 1977).…”

Ligation of RNA-Containing Duplexes by Vaccinia DNA Ligase

“…Remarkably, the T4 DNA ligase was not active in joining a 3′-OH DNA to a 5′-phosphate RNA molecule; i.e., 3′-OH poly(dA) was not ligated to 5′-32 P-poly(rA), and 3′-OH poly-(dT) was not joined to 5′-32 P-poly(rU). Similarly, an early study of ligation of homopolymers by a DNA ligase from mouse L cells (this activity presumably corresponds to DNA ligase I) showed that the enzyme catalyzed the joining of 3′-OH RNA to 5′-phosphate DNA on a DNA template, but did not catalyze ligation of 3′-OH DNA to 5′-phosphate RNA (Bedows et al, 1977).…”