Competition between the RNA transcript and the nontemplate DNA strand during R-loop formation in vitro: a nick can serve as a strong R-loop initiation site

Abstract

Upon transcription of some sequences by RNA polymerases in vitro or in vivo, the RNA transcript can thread back onto the template DNA strand, resulting in an R loop. Previously, we showed that initiation of R-loop formation at an R-loop initiation zone (RIZ) is favored by G clusters. Here, using a purified in vitro system with T7 RNA polymerase, we show that increased distance between the promoter and the R-loop-supporting G-rich region reduces R-loop formation. When the G-rich portion of the RNA transcript is downstream from the 5' end of the transcript, the ability of this portion of the transcript to anneal to the template DNA strand is reduced. When we nucleolytically resect the beginning of the transcript, R-loop formation increases because the G-rich portion of the RNA is now closer to the 5' end of the transcript. Short G-clustered regions can act as RIZs and reduce the distance-induced suppression of R-loop formation. Supercoiled DNA is known to favor transient separation of the two DNA strands, and we find that this favors R-loop formation even in non-G-rich regions. Most strikingly, a nick can serve as a strong RIZ, even in regions with no G richness. This has important implications for class switch recombination and somatic hypermutation and possibly for other biological processes in transcribed regions.

FIG. 1.

Effect of the nonhybridizing portion of the RNA transcript on R-loop formation. (A) Linearized pDR51 (containing three Sγ3 repeats downstream of the T7 promoter) and pDR72 (containing three Sγ3 repeats separated from the T7 promoter by one repeat length of random sequence) were either mock transcribed (lanes 1 and 11 for pDR51 and lanes 6 and 16 for pDR72) or transcribed with T7 RNA polymerase in the presence of [α-³²P]UTP. The samples were treated with RNase A after either transcription (lanes 2 to 4 and 7 to 9 for pDR51 and pDR72, respectively) or during transcription (lanes 12 to 14 and 17 to 19 for pDR51 and pDR72, respectively). The fifth lane in each set is a transcribed sample treated with RNase A and RNase H1 (lanes 5, 10, 15, and 20). The top panel is the ethidium bromide-stained gel profile. The positions of the linear switch substrate-containing fragments are marked “L.” R-loop molecules run slower than the linear fragments and are seen as a shifted band (marked “Shift”) running higher than the linear position. The shifted band is not present in the RNase H-treated lane because the RNA within the R loop is nucleolytically removed by RNase H. The bottom panel shows the [α-³²P]UTP radiolabel exposure of the same gel shown in the top panel. Most of the radiolabel localizes with the shifted bands and is not seen with the linear fragments or with the mock-transcribed or the RNase H-treated samples at either position. (B) Model for R-loop formation at the G-rich DNA in pDR72 upon removal of the nonhybridizing portion of the transcript in the presence of RNase A. The approximate positions of the T7 promoter (black arrow), the three Sγ3 switch repeats (three solid arrows showing the C-rich repeats on the template strand DNA, complementary to the G-rich nontemplate strand), and the one-repeat long random sequence DNA between the promoter and the start of the switch repeats (striped line) in pDR72 are shown. The corresponding non-G-rich portion of the RNA is shown as an open line, followed by the G-rich part of the transcript in the switch region, shown as a solid gray line. In the presence of RNase A during transcription, the non-G-rich part of the transcript is preferentially digested (dashed region) compared to the G-rich part of the transcript because RNase A cuts after Cs and Us in the RNA. This allows for increased mobility of the G-rich portion of the transcript (downward arrow), and it can now establish an RNA:DNA hybrid.

FIG. 2.

Presence of G clusters at a distance upstream of the switch repeats enhances R-loop formation. (A) The substrates are represented by the line figure and show the presence of RIZ motif A (two four-G clusters), C (one four-G cluster), or B (no G clusters) as gray blocks in pDR72A, pDR72C, or pDR72B, respectively. The motif (A, C, or B) is placed downstream of the T7 promoter. The three Sγ3 switch repeats (solid black arrows) located downstream are separated from the motif by a one-repeat long random sequence (shown as a striped line). Linearized substrates were transcribed and run on agarose gels to assess the presence of shifted species in the transcribed samples treated with RNase A after transcription but not with RNase H1. The top panel shows the ethidium bromide-stained gel. Lanes 1 to 5 have pDR72A, lanes 6 to 10 have pDR72C, and lanes 11 to 15 have pDR72B. Lanes 1, 6, and 11 contain the mock-transcribed samples for pDR72A, pDR72C, and pDR72B, respectively. The next three lanes in each set contain T7-transcribed substrates treated with RNase A afterwards. The last lanes in each set contain transcribed sample treated with RNase A and RNase H1 afterwards. The position of the linearized fragment containing the switch region is marked “L.” The position of the transcription-induced shifted species, seen in lanes 2 to 4 for pDR72A, 7 to 9 for pDR72C, and 12 to 14 for pDR72B, is marked “Shift.” The bottom panel shows the radiolabel densities at the “Shift” positions. (B) Model of R-loop formation at REZ switch repeats in the presence of G-clustered RIZs located at a distance upstream of the REZ. The top panel shows the initial phase of R-loop formation, where the short but stable nucleating RNA:DNA hybrid forms at the G-clustered RIZ motif. The middle panel demonstrates the next step, when an R loop is established at the G-rich switch repeats (REZ). This process is more efficient in the substrates that have the G-clustered RIZ motifs at the distant upstream region. Because a G-rich REZ is not present near the RIZ motifs, the short RNA:DNA hybrid formed at the G-clustered RIZ dissociates relatively quickly (bottom panel) compared to regions where RIZ and REZ are close to each other. The R loop formed at the downstream REZ persists longer because of better stabilization.

FIG. 3.

The location and extent of the RNA:DNA hybrid region vary between linearized and negatively supercoiled substrates. The top line represents the length of pDR72A tested for the presence of R loops. The T7 promoter is shown as an elevated arrow above the line. The solid black box represents the location of motif A (two four-G clusters), followed by the open box representing the one-repeat length random sequence upstream of the three Sγ3 repeats shown as three black arrows. The next line shows all the Cs present in the region sequenced, and each short vertical line represents a C that can potentially be converted to T upon sodium bisulfite treatment (if unpaired) following transcription. The asterisk shows the location of the methylated C in a dcm site CC^met(A/T)GG, where the methylated second C escapes bisulfite modification. The light gray region covers the Cs present in motif A and the random sequence, whereas the darker gray region covers the Cs in the Sγ3 repeat regions. Each of the horizontal lines below the full display of Cs represents an observed R-loop molecule, with each short vertical line representing an observed C-to-T conversion. (A) The linearized and transcribed pDR72A molecules containing R-loop structures are shown. R loops on the 20 linearized molecules shown are mostly contained within the switch repeats, and there are only rare conversions near the promoter or the A motif, none of which are continuous with the single strandedness at the switch repeat regions. Two molecules (10%) show extension of the single strandedness downstream of the switch repeats. (B) In contrast to the linearized molecules (A), out of 29 supercoiled molecules with R-loop structures, 7 (24%) start near the promoter or the G-clustered A motif. Four (∼14%) show 25-nt or longer stretches of conversion around the promoter/motif region. (C) A model of R-loop formation at a negatively supercoiled substrate with the RIZ and REZ separated by a non-G-rich sequence. In the supercoiled version of pDR72A, where the G-clustered RIZ (two four-G repeats) is separated from the REZ (three Sγ3 repeats), the R loops for many molecules begin upstream of the REZ and are continuous from the RIZ to the REZ. This pattern is different from that of the linearized molecules. Negative supercoiling favors transient separation of DNA strands and is shown by a solid black arrow pointing upwards. This gives the non-G-rich portion of the transcript an increased opportunity to compete (gray downward arrow) with the nontemplate DNA strand for RNA:DNA hybrid formation.

FIG. 4.

The presence of a nick on the nontemplate strand downstream of the promoter increases R-loop formation at the downstream switch repeats. Three substrates, pDR18, pDR87, and pDR88, were mock incubated or incubated with the nicking enzyme Nt.BbvCI and then linearized with SalI. pDR87 and pDR88 are derived from pDR18, and all have four Sγ3 repeats downstream of the promoter, except that pDR18 does not have a BbvCI recognition site. A nicking site (shown as an inverted triangle) is present upstream of the promoter in pDR87 and between the promoter and the switch repeats in pDR88. The four arrows represent the four Sγ3 repeats. For each substrate, the first three lanes are unnicked substrates, whereas the next three lanes contain the same substrate treated with Nt.BbvCI. The position of the linearized fragment is marked “L.” In each subset of lanes, the first lane is mock-transcribed substrate, followed by a T7-transcribed substrate subsequently treated with RNase A after transcription. The third lane contains the transcribed substrate treated with RNase A and RNase H. All samples were organically extracted before being run on an agarose gel. The top panel is the ethidium bromide-stained profile of the agarose gel. Comparable amounts of shifted nucleic acids are seen for pDR18, pDR87, or the unnicked version of pDR87. The nicked version of pDR88 shows a much larger amount of shift, indicating a highly efficient R-loop formation compared to unnicked substrates or to a substrate with a nick upstream of the promoter. The bottom panel shows the same gel with the location of radiolabeled ([α-³²P]UTP) bands. While almost no radiolabel is present at the position where the switch repeat-containing linearized fragments run, we observed the presence of a large amount of radiolabel at the shifted position for nicked and transcribed pDR88 (compared to the shifted positions in other substrates and unnicked pDR87). It should be noted that this shifted position, as also observed in the top panel, runs slower than the shifted positions in other substrates, and this mobility change could be because of the presence of the partially displaced nontemplate DNA strand. In the same lane (lane 19), an additional radiolabeled band localizes near the linear fragment (marked “L”), indicating that short RNA:DNA hybrids are formed at or near the nicked position.

FIG. 5.

Model of R-loop formation on substrates containing a nontemplate strand nick upstream or downstream of the promoter. The model in the top panel shows the mechanism of R-loop formation when the nick on the nontemplate DNA strand is present upstream of the promoter. The nick position is shown as a discontinuity on the nontemplate strand. During transcription, the elongating RNA polymerase unwinds the incoming duplex DNA, but the upstream edge of the transiently open transcription bubble upstream of the RNA polymerase closes and forms duplex DNA relatively quickly (the solid DNA_ON downward arrow depicts a relatively strong DNA duplex formation propensity, whereas a dashed upward arrow depicts a weaker DNA dissociation propensity). This makes the RNA (the gray strand) less efficient in establishing an RNA:DNA hybrid. The model in the bottom panel shows the mechanism of R-loop formation when the nick on the nontemplate DNA strand is present downstream of the promoter. As the transcribing RNA polymerase approaches the nick and unwinds the duplex DNA, the template strand enters the RNA polymerization active site whereas the nontemplate strand is displaced because it loses its contact with the duplexed upstream DNA (due to the presence of the nick, which reduces the DNA duplex formation propensity [dashed black downward arrow showing relatively reduced DNA_ON propensity] and increases the chances of the template and the nontemplate DNA strand to remain single stranded (solid upward DNA_OFF arrow]). This gives the nascent RNA coming out of the RNA exit pore in the RNA polymerase a greater chance to establish an RNA:DNA hybrid with the DNA template strand (downward gray arrow showing an increase in RNA_ON).

FIG. 6.

Presence of a nick on the nontemplate strand downstream of the promoter increases R-loop formation even in the absence of switch repeats. Three substrates, pDR16, pDR89, and pDR90, were mock incubated or incubated with the nicking enzyme Nt.BbvCI, and then linearized with SalI. The position of the linearized fragment is marked “L.” No switch repeats are present in these substrates. pDR16 does not have an Nt.BbvCI recognition site, whereas a nicking site (shown as an inverted triangle) is present upstream of the promoter in pDR88 and downstream of the promoter in pDR90. In each set, the first three lanes are unnicked whereas the next three lanes contain the same substrate treated with Nt.BbvCI. In each subset of lanes, the first lane is mock-transcribed substrate, followed by a lane with T7-transcribed substrate treated with RNase A after transcription. The third lane contains the transcribed substrate treated with RNase A and RNase H. The top panel is the ethidium bromide-stained gel showing the positions of the linearized fragments containing the T7 promoter. No shifts are seen for pDR16, pDR89, or the unnicked version of pDR90. Although small in amount, the nicked version of pDR90 shows a shifted band, indicating an increased ability of nicked pDR90 to form R loops compared to unnicked pDR16, pDR89, and pDR90 or with a promoter-upstream nick in pDR89. The bottom panel is the same gel showing the location of radiolabeled bands (labeled with [α-³²P]UTP). A distinct radiolabeled band is observed at the shifted position for nicked and transcribed pDR90, compared to the shifted or the linear (“L”) positions for other substrates, where no radiolabel localization is seen. In the same lane (lane 19), an additional radiolabeled band localizes near the linear fragment, indicating an increased propensity for RNA:DNA hybrid formation by the transcript in the presence of the nick.

FIG. 7.

A nontemplate strand nick downstream of the promoter increases R-loop formation more when the nick is immediately upstream of the switch repeats. (A) Two substrates, pZZ6 and pZZ7, were mock incubated or incubated with the nicking enzyme Nt.BbvCI and then linearized with SalI before transcription. pZZ6 and pZZ7 are similar and have three Sγ3 repeats present, with a one-repeat long random sequence spacer DNA between the promoter and the start of the switch repeats. The nontemplate strand nicking site (shown as an inverted triangle) on pZZ6 is present at the start of the one-repeat long random spacer sequence, immediately downstream of the promoter between positions +9 and +10. pZZ7 has a nicking site at the end of the one-repeat long random spacer downstream of the promoter (nick site located between positions +69 and +70) and immediately upstream of the start of the switch repeats. The position of the linearized fragment is marked “L.” For each substrate, the first three lanes contain unnicked samples, whereas the next three lanes contain the same substrate treated with Nt.BbvCI. In each subset of three lanes, the first lane contains a mock-transcribed substrate, whereas the second lane contains a T7-transcribed sample treated with RNase A after transcription. The third lane contains transcribed substrate treated with RNase A and RNase H after transcription. The top panel is the ethidium bromide-stained gel showing the positions of the linearized fragments (“L”) containing the T7 promoter. Shifted species were seen for nicked and transcribed substrates (lanes 5 and 12 for pZZ6 and pZZ7, respectively) but not for unnicked and transcribed pZZ6 or pZZ7 (lanes 2 and 9). Nicked pZZ6 showed a much stronger shift than nicked pZZ7 (compare lane 5 with lane 12). The shifted species were sensitive to RNase H (lanes 3, 6, 10, and 13). Different bands seen in the shifted species are likely different conformational variants of the R-loop structure formed with the displaced nontemplate strand DNA. Although the shift seen for nicked and transcribed pZZ7 (lane 12) is weaker than that in the corresponding lane for pZZ6 (lane 5), it is stronger than that in the corresponding lane for unnicked pZZ6 or pZZ7 (lanes 2 and 9, respectively), demonstrating that the presence of a promoter-downstream nick can help in R-loop formation, even if it is located at a distance from the promoter. The bottom panel shows the location of radiolabeled ([α-³²P]UTP) bands in the same gel. The shifted positions in lanes 5 and 12, as deduced from panel A, are radiolabeled. While no radiolabel is present at the position where the linearized fragments migrate (marked “L”) for the unnicked versions of the substrates, radiolabeled bands at this position for nicked and transcribed pZZ6 and pZZ7 are observed. (B) Model of R-loop formation with promoter-downstream nicks present at various locations. The top panel shows the mechanism of R-loop formation in a nicked substrate (pZZ6) when the nick is present immediately downstream of the promoter and one repeat length upstream of the three Sγ3 repeats (three open arrows show the positions of the switch repeats). When the RNA polymerase passes through the nick, the nontemplate strand is displaced from the duplex form of DNA. Since the nick is close to the promoter and transcription start site, the length of the displaced, untethered nontemplate strand DNA behind the transcribing RNA polymerase is comparable to the length of the transcript coming out of the exit pore of the RNA polymerase, resulting in comparable propensities for binding with the template DNA strand (shown as downward DNA_ON and RNA_ON arrows for the nontemplate DNA and the RNA, respectively). However, the stability of the RNA:DNA duplex is greater than that for DNA:DNA, making an RNA:DNA hybrid more likely in the nicked substrates, even in random non-G-rich regions. This initial nucleation, though transient, helps in further establishment of R loops at the downstream G-rich switch repeats. The bottom panel represents the mechanism of R-loop formation when the nontemplate strand nick is present 60 nt further downstream of the promoter but immediately upstream of the start of the switch repeats (pZZ7). When the RNA polymerase reaches the nick position, the transcript length is already over a repeat long with a mostly non-G-rich (and therefore nonhybridizing) sequence composition. Free RNA is longer than untethered nontemplate strand DNA downstream of the nick and upstream of the RNA polymerase, resulting in higher molecular mobility of the DNA end and ensuring a higher frequency of collision between the DNA strands compared to the internal G-rich region of the transcript. This is shown as a dashed (weaker) RNA_ON arrow. Despite a thermodynamic stability advantage, any RNA:DNA hybrid formation is suppressed because of unfavorable access of the G-rich portion of the transcript to the template strand. However, even with these factors opposing R-loop formation, nicked pZZ7 forms R loops more than unnicked pZZ6 or unnicked pZZ7 (see panel A). This shows that the presence of a nick offers the RNA an increased opportunity to compete with the nontemplate strand DNA.