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

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.