On the Initiation of Mammalian RNA Polymerase at Single-Strand Breaks in DNA

Abstract

Mammalian RNA polymerase B is able to initiate at single-strand breaks in the DNA template. A 3'-OH end at a nick is required for initiation whereas the 5'-end may be either -OH or phosphate. The 3'-OH group does not function as a primer. An appreciable part of the newly synthesized RNA started at a nick remains associated with the DNA in hydrid form. Initiation of exogeneous RNA polymerase B on chromatin exhibits similar requirements.The recognition of initiation sites on the DNA by RNA polymerase poses a major problem for the understanding of the process of transcription. Intact mammalian DNA is a poor template for mammalian polymerase B [2] (for a review see [I]). Treatment of the DNA with DNAase I greatly improves its template activity [3].Since mammalian RNA polymerase B is known to attach preferentially to single-stranded DNA [4,5] it is possible that the increased activity of DNAasetreated DNA results from the generation of singlestranded regions. Permanent single-stranded regions could arise from extensive degradation or, alternatively, transitory single-stranded regions could arise opposite single-strand breaks (nicks) by partial or temporary destabilization of double-stranded structure [6,7]. Such a destabilization may also facilitate strand separation required for the initiation process A third possibility is that the polymerase recognizes the free ends at an internal nick. It may then be possible that a specific configuratiori at the nick site is required for the initiation event.We have investigated this question by studying the effect on the template activity of modifications of the DNA by different enzymic treatments. Methods used to modify the DNA are outlined in Table 1. As can be seen these methods allow the preparation of DNA samples containing single-strand nicks with different chemical structures.

Three-dimensional model of yeast RNA polymerase I determined by electron microscopy of two-dimensional crystals.

“…Templates 1 and 2 were respectively 20 and 41 nucleotides long, double-stranded DNA with an internal gap of one nucleotide at positions 9 and 18 respectively. The gaps were shown to be recognized as an initiation site by calf thymus RNA polymerase II (Dreyer and Hausen, 1976). The coding 3' end of DNAs 3 and 4 present a 9 nucleotide long, single-stranded poly(C) extension and a double-stranded portion respectively 11 and 29 nucleotides long.…”

“…Thus transcription would proceed from groove to channel. The synthetic DNAs used in this study are likely to be specifically bound and oriented in the enzyme since both single-stranded breaks (Dreyer and Hausen, 1976) and single-stranded-double-stranded junctions in 3' poly(C) extended DNAs (Kadesch and Chamberlin, 1982) (Metzger et al, 1989 (Darst et al, 1991). However, the authors had to take into account that channel and groove cannot bind 73 bp and that additional DNA has to interact with the enzyme surface.…”

Inhibition of mammalian RNA polymerase by 5,6-dichlororibofuranosylbenzimidazole (DRB) and DRB triphosphate

“…All other materials were as previously described (8,14). RESULTS Effect of DRB triphosphates on isolated polvmerases.…”

Transcription of rat liver deoxyribonucleic acid in vitro at low ionic strength

“…was 4.6 x 106 in native form, and 2 x IO' when denatured. A fraction of this DNA preparation was treated with nuclease SI in low salt concentration as described by Dreyer & Hausen (1976), specifically to destroy DNA strands opposite nicks, converting these into double-stranded ends. Another fraction of this DNAase-treated DNA was treated with nuclease SI in high salt concentration to digest the singlestranded sequences (Vogt, 1973).…”