Name: Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase (Video) | JoVE Journal
Uploaded: 2010-04-29T17:00:00.000+00:00
Duration: 7 min 27 s
Description: Learn how to study replication fork restart after collision with head-on RNA polymerase using biotinylated DNA and magnetic bead immobilization. This method reveals how Mfd facilitates replisome displacement and DNA replication resumption.

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We are especially grateful to Seth Darst for providing RNAP and Mfd proteins and expression vectors. We are also thank Sergei Borukhov for providing GreB and Dan Zhang for technical support. This work was supported by a grant from the National Institutes of Health (M.O.D.) and by a Marie-Josee and Henry Kravis Fellowship at the Rockefeller University (R.T.P.).

This method was used in the research reported in <a href="http://www.sciencemag.org/cgi/content-nw/full/327/5965/590/F3">Pomerantz and O'Donnell, Science 327, 590-592 (2010)</a>.
1. Assembly of a halted RNAP elongation complex was performed as follows:
500 nM final concentration of E. coli RNAP σ70 HE was mixed with 5 nM final concentration of a biotinylated 3.6 kb DNA template containing the T7A1 promoter in 100 μl of buffer A (20 mM Tris-Cl (pH 7.5), 8 mM MgCl2, 0.5 mM EDTA, 5 mM DTT, 10% glycerol) for 10 min at 37 °C. 100 μM of ApU and 40 μM each of GTP, ATP, and CTP were added for an additional 10 min at 37 °C.
2. Immobilization of the halted RNAP elongation complex onto streptavidin beads was performed as follows:
200 μl of streptavidin magnetic coated beads (Invitrogen) were added to the reaction above for 10 min at room temp.
3. Purification of the immobilized halted RNAP elongation complex was performed as follows:
The beads (from above) were washed 5 times with 0.9 ml of buffer A containing 0.75 M NaCl, 200 μg/ml heparin, and 20 μg/ml ssDNA (The supernatant was removed after each wash step by magnetic separation.) Next, the beads were washed 2 times with 0.9 ml of buffer A.
4. Formation of a replication fork downstream relative to the head-on RNAP was performed as follows:
The beads (from above) were resuspended in 100 μl of New England Biolabs buffer 4 and 10 units of Sap I (New England Biolabs) was added for 10 min at 37 °C. The beads were washed 3 times with 0.9 ml of buffer A and then resuspended in 50 μl of Quick T4 Ligation reaction buffer (New England Biolabs). 2 μl of Quick T4 ligase (New England Biolabs) was added along with 6 nM final concentration of pre-annealed forked DNA (RP10, RP22, RP25) for 10 min at room temp. The beads were washed 3 times with 0.9 ml of buffer A. 
5. Assembly of the replisome and initiation of leading strand synthesis at the replication fork was performed as follows:
448 pmol of DnaB (as hexamer) was incubated with the beads 150 μl of buffer A (20 mM Tris-Cl (pH 7.5), 8 mM MgCl2, 0.5 mM EDTA, 5 mM DTT, 10% glycerol) for 30 s at 23 °C. 4.9 pmol of Pol III* (Pol III HE minus β), 15 pmol of β, 2 mM ATP, and 60 μM each of dGTP and dATP were added to a volume of 200 μl and incubated a further 5 min at 37 °C. Replication was initiated upon adding 10 μg SSB and 24 μM each of dATP and dTTP along with [α-32P]dTTP and [α-32P]dCTP (specific activity, 3,000-5,000 cpm/pmol) to a final volume of 250 μL. Reactions were terminated after 10 min upon adding 12 μl of 500 mM EDTA.
6. Purification and concentration of the radio-labeled DNA products was performed as follows:
The beads (from above) were boiled after the reaction was terminated in order to remove the DNA from the beads. Any residual DNA remaining attached to the beads was removed by treating the beads with proteinase K in a volume of 10 ul for 30 min at 50 °C. The beads were then boiled and the supernatant was removed by magnetic separation. The combined supernatant containing the DNA was purified using the Qiagen PCR Cleanup kit. Purified radio-labeled DNA products were then analyzed in an alkaline agarose gel.
*Replication in the absence of a head-on RNAP halted elongation complex was performed as above, however, σ70 was omitted.
Representative Results:
Collision of the replisome with a head-on RNAP usually results in two products of 2.5 kb and 3.6 kb in length. The 2.5 kb product represents the length of the DNA from the fork to the halted RNAP and results from replisome stalling upon collision with the RNAP. The 3.6 kb product represent full-length DNA and results from either incomplete occupancy of the promoter by RNAP or partial replisome read-through of the head-on RNAP. A good or accurate result demonstrates that approximately 50% full-length DNA is produced. However, in some cases less  occupancy of the promoter by RNAP occurs and a higher percentage of full-length DNA is observed since a greater population of replisomes do not encounter a head-on RNAP. Replication in the absence of a halted RNAP results in only full-length DNA (3.6 kb).
<img src="/files/ftp_upload/1919/1919fig1.jpg" alt="DNA replication process; diagram with RNAP block, replisome stalling, full-length DNA replication." data-altupdatedat="1761735611000" data-alt="Figure 1"> Figure. 1 The replisome is impeded by a head-on RNAP. (A) Experimental setup. A E. coli RNAP halted elongation complex was assembled on a biotinylated DNA containing the T7A1 promoter as previously described (9). Briefly, a halted elongation complex was formed 20 bps downstream from the promoter by incubating RNAP holoenzyme with biotinylated DNA for 10 min followed by the addition of ApU, GTP, ATP, and CTP for a further 10 min. Streptavidin beads were added for an additional 10 min then the beads were washed five times each with 0.9 ml of buffer containing 0.75M NaCl, 200 mg/ml heparin, and 100 nM single-strand DNA to remove unstable RNAP-DNA complexes and excess NTPs. The DNA was then digested with SapI, washed, and ligated to a pre-annealed forked DNA. The beads were washed again prior to assembly of the replisome. The promoter is located 2.5 kb from the fork and is oriented to direct transcription towards the fork. (B) DnaB was added then the replisome was assembled and replication was initiated either in the presence (lane 2) or absence (lane 1) of a head-on RNAP.

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Washing the halted elongation complex bound to the beads with high salt is critical in order to remove any unstable or non-specifically bound RNAP molecules. RNAP has an extremely high affinity for primer-template type DNA structures. Thus, it is necessary to remove such non-specific RNAP-DNA complexes in order to perform replication from the primer-template. Concentrating the radio-labeled DNA products using the Qiagen PCR purification kit is also critical in order to obtain a high enough radioactive signal that can be observed in the gel.

<table><tbody><tr><td>Dynabeads M-270</td><td>Invitrogen</td><td>653.05</td><td></td></tr><tr><td>SapI</td><td>New England Biolabs</td><td>R0569S</td><td></td></tr><tr><td>T4 Quick Ligase</td><td>New England Biolabs</td><td>M2200S</td><td></td></tr><tr><td>PCR Purification Kit</td><td>Qiagen</td><td>28104</td><td></td></tr></tbody></table>

direct restart of a replication fork stalled by a head-on rna polymerase

Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase

In vivo studies suggest that replication forks are arrested due to encounters with head-on transcription complexes. Yet, the fate of the replisome and RNA polymerase (RNAP) following a head-on collision is unknown. Here, we find that the E. coli replisome stalls upon collision with a head-on transcription complex, but instead of collapsing, the replication fork remains highly stable and eventually resumes elongation after displacing the RNAP from DNA. We also find that the transcription-repair coupling factor, Mfd, promotes direct restart of the fork following the collision by facilitating displacement of the RNAP. These findings demonstrate the intrinsic stability of the replication apparatus and a novel role for the transcription-coupled repair pathway in promoting replication past a RNAP block.

The fate of the replisome following a collision with a head-on RNA polymerase (RNAP) is unknown. We find that the replisome stalls upon collision with a head-on RNAP, but resumes elongation after displacing the RNAP from DNA. Mfd promotes replication restart by facilitating displacement of the RNAP after the collision.

In vivo studies suggest that replication forks are arrested due to encounters with head-on transcription complexes. Yet, the fate of the replisome and RNA ...

The overall goal of this procedure is to observe the fate of the Repli zone and RNA polymerase or RNAP following a head-on collision. This is accomplished by first assembling the transcription complex on biotinylated DNA and immobilizing it on Stripp tablet and beads, which will result in the halted RNAP elongation complex. The second step of the procedure is to ligate preen pre-need forked DNA to the RNAP elongation complex to form a replication fork downstream of the head-on RNAP.The third step of the procedure is to assemble the repli zone and initiate leading strand synthesis at the replication fork. The final step of the procedure is to isolate the DNA from the magnetic beads and purify the DNA over a PCR cleanup column. Ultimately, results can be obtained that show the roso stalls upon collision with the head-on transcription complex through an alkaline agro gel of the purified radio labeled DNA products.Hi, I'm Richard Pomerance from the Laboratory of Mike O'Donnell at the Rockefeller University. Today I'll show your procedure how to perform a collision of the ribosome with a head on RNA polymerase in solid phase. I use this procedure in our laboratory to study how the process of replication is affected by transcription complexes along DNA.So let's get started. To begin this protocol mix, RNAP holo enzyme with a biotinylated 3.6 KB DNA template containing the T seven A one promoter in 100 microliters of buffer a incubate the mixture for 10 minutes at 37 degrees Celsius. After the 10 minute incubation, add ribonucleotides adenosine, tri phosphate, cytidine phosphate, and Aden allele uridine, which will limit RNA synthesis to 20 nucleotides.Incubate the solution for an additional 10 minutes at 37 degrees Celsius to immobilize the halted RN ape elongation complex onto beads. Add the solution to streptavidin coated magnetic beads. Let the mixture incubate for 10 minutes at room temperature.Purify the immobilized halted RNAP elongation complex by washing the beads with 0.9 milliliters of high salt buffer. To remove non-specific R-N-A-P-D-N-A complexes following each wash, remove the supernatant by magnetic separation. This wash should be repeated five times.Then the beads should be washed twice more with 0.9 milliliters of buffer A.After the final wash, the downstream replication fork can be assembled to form a replication fork downstream of the head-On RNAP Resus, suspend the magnetic strip to evident beads attached to the halted RNAP elongation complex in 100 microliters of New England Biolabs buffer four. Next, add 10 units of shrimp alkaline phosphatase or SAP one, and incubate the sample for 10 minutes at 37 degrees Celsius. Wash the beads three times with 0.9 milliliters of buffer A.Then resuspend the beads in 50 microliters of quick T four ligation reaction buffer.Add two microliters of quick T four ligase and pre-need forked DNA, incubate the solution for 10 minutes at room temperature. Finally, wash the beads three more times with 0.9 milliliters of buffer A mix hexamer, DNAB helicase with the magnetic strep A and beads which are attached to the halted RNAP elongation complex and the downstream replication fork. Prepare the solution in 150 microliters of buffer A and incubate it for 30 seconds at 23 degrees Celsius.Following the brief 32nd incubation at 23 degrees Celsius. At polymerase three beta clamp, A-T-P-D-G-T-P-D-A-T-P alpha P 32 labeled DGTP and alpha P 32 labeled DATP to a volume of 200 microliters. Incubate the sample for five minutes at 37 degrees Celsius.Initiate replication by adding single-stranded DNA binding protein DCTP and DTTP. Also add alpha P 32, labeled DTTP and DCTP to a final volume of 250 microliters. After 10 minutes, terminate the reactions by adding 12 microliters of 0.5 molar EDTA.Next, boil the strp din beads to release the DNA from the beads. Remove any residual DNA by treating the beads with proteinase K for 30 minutes at 50 degrees Celsius. Boil the beads again and remove the supernatant by magnetic separation.Purify the combined supernatant containing the DNA using the Kyogen PCR cleanup kit. Finally, analyze the purified radio label DNA products in an alkaline aros gel Collision of the zone with a head on RNAP usually results in two products of 2.5 KB and 3.6 KB in length. The 2.5 KB product represents the length of the DNA from the fork to the halted RNAP.This results from repli stalling upon collision with the RNAP. The 3.6 KB product represents full length DNA and results from either incomplete occupancy of the promoter by RNAP or partial repli readthrough of the head-on RNAP. A good result demonstrates that approximately 50%full length DNA is produced.However, in some cases, less occupancy of the promoter by RNAP occurs and a higher percentage of full length DNA is observed. This is because a greater population of repli zones does not encounter a head on RNAP replication in the absence of a halted RNAP results in only full length DNA, We've just showed you how to perform replication and solid phase in the presence of a halted RNA polymerase elongation complex bound to the DNA. When doing this procedure, it's important to wash the halted transcription complex with high salt to remove excess RNA polymerase, which non-specifically binds to the DNA and inhibits replication.So that's it. Thanks for watching and good luck with your experiments.

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Learn how to study replication fork restart after collision with head-on RNA polymerase using biotinylated DNA and magnetic bead immobilization. This method reveals how Mfd facilitates replisome displacement and DNA replication resumption.

Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase

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