Transcription regulatory elements are punctuation marks for DNA replication

Abstract

Collisions between DNA replication and transcription significantly affect genome organization, regulation, and stability. Previous studies have described collisions between replication forks and elongating RNA polymerases. Although replication collisions with the transcription-initiation or -termination complexes are potentially even more important because most genes are not actively transcribed during DNA replication, their existence and mechanisms remained unproven. To address this matter, we have designed a bacterial promoter that binds RNA polymerase and maintains it in the initiating mode by precluding the transition into the elongation mode. By using electrophoretic analysis of replication intermediates, we have found that this steadfast transcriptioninitiation complex inhibits replication fork progression in an orientation-dependent manner during head-on collisions. Transcription terminators also appeared to attenuate DNA replication, but in the opposite, codirectional orientation. Thus, transcription regulatory signals may serve as ''punctuation marks'' for DNA replication in vivo.collisions ͉ promoter ͉ terminator I mpairment of DNA replication is believed to be a major factor in genomic instability (1-13). Because transcription and replication share the same template, occasional collisions between the two machineries are inevitable and can interfere with replication fork progression. Collisions between the elongating RNA polymerase and the replication fork have been well documented in vitro (14-17) and in vivo in both Escherichia coli (18)(19)(20) and Saccharomyces cerevisiae (6,21,22). The consensus from those studies was that head-on collisions with elongating RNA polymerase are much more detrimental for replication fork progression than codirectional collisions. Although it was suggested that replication stalling during the head-on collisions with transcription was caused by topological stress in the DNA separating the two machineries (18,19,22,23), we have recently shown that it is caused by their direct, physical interaction (20). These results, combined with the data on preferred codirectional alignment of transcription units with the direction of replication in prokaryotes (23-27), have led to the suggestion that the main disadvantage of the head-on collisions could be their inhibitory effect on DNA replication.All of the experimental studies cited in the preceding paragraph evaluated the effects of elongating RNA polymerase on the progression of the replication fork. Is there an interplay between the replication machinery and the transcriptioninitiation complex? To the best of our knowledge, there have been few studies on this matter. One intriguing example was the detection of a polar replication fork pause site at the tRNA locus of S. cerevisiae, which depended on the functionality of both the promoter and the RNA polymerase III (pol III) (22). It was believed that the replication fork was attenuated during the encounter with the elongating RNA polymerase (22). A later study, however, suggest...

Genome-wide coorientation of replication and transcription reduces adverse effects on replication in Bacillus subtilis

“…The same DNA template is used by RNA polymerase (RNAP) for transcription and by the replisome (DNA polymerase and associated proteins) for replication. Transcription complexes that are stalled, initiating, or terminating can slow or block replication (8,9). RNAP and the replisome can collide when moving toward each other (head-on), or when the replisome, which moves faster than RNAP, catches up with RNAP moving in the same direction (codirectional).…”

Polymerase trafficking

“…20 Notably, GreB has previously been shown to prevent replication fork arrest due to an encounter with a transcription termination complex, which further supports a role for transcription factors in reducing conflicts between replication and transcription. 30 These studies indicate that proper transcription elongation is necessary for transcription regulation and repair that is necessary for ensuring complete DNA replication in the face of highly stable transcription complexes. Since obstructing replication leads to mutagenesis and chromosomal rearrangements, transcription factors that prevent replication fork arrest likely play an important role in maintaining genome integrity.…”

“…Yet, it is important to note that under certain conditions co-directional transcription complexes also arrest the fork; R-loops and RNAP backtracking contribute to this effect. 30,32 We favor a model whereby deficient transcription elongation leads to the accumulation of replication fork blocking RNAP arrays (Fig. 2, right).…”

Analysis of Chromosomal Replication Progression by Gel Electrophoresis

“…Later on, it was adapted to study the DNA replication intermediates (RIs) (Brewer & Fangman, 1987). Since then, 2D agarose gel electrophoresis was used to map and characterize replication origins (Brewer & Fangman, 1988;Gahn & Schildkraut, 1989;Liu & Botchan, 1990;Schvartzman et al, 1990;Linskens & Huberman, 1990 b;Friedman & Brewer, 1995;Bach et al;, to analyze the progression of DNA replication along a DNA fragment (Azvolinsky et al, 2006), to characterize replication fork barriers (Brewer & Fangman, 1988;Linskens & Huberman, 1988;Hernandez et al, 1993;Wiesendanger et al, 1994;Samadashwily et al, 1997, López-Estraño et al, 1998, Possoz et al, 2006Mirkin et al, 2006, Boubakri et al, 2010, replication termination (Zhu et al, 1992;Santamaría et al, 2000a,b), origin replication interference (Viguera et al, 1996), RIs knotting (Viguera et al, 1996;Sogo et al, 1999), fork reversal Fierro-Fernandez et al, 2007a) or the topology of partially replicated plasmids (Martín-Parras et al, 1998;Lucas et al, 2001). See (Schvartzman et al, 2010) for an excellent review in plasmid DNA replication analyzed by 2D-gel.…”