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Direct inhibition of RNA polymerase II transcription by RECQL5.

Aygün O, Xu X, Liu Y, Takahashi H, Kong SE, Conaway RC, Conaway JW, Svejstrup JQ - J. Biol. Chem. (2009)

Bottom Line: Here, we show that RECQL5 inhibits both initiation and elongation in transcription assays reconstituted with highly purified general transcription factors and RNAPII.Such inhibition is not observed with the related, much more active RECQL1 helicase or with a version of RECQL5 that has normal helicase activity but is impaired in its ability to interact with RNAPII.Indeed, RECQL5 helicase activity is not required for inhibition.

View Article: PubMed Central - PubMed

Affiliation: Mechanisms of Transcription Laboratory, Clare Hall Laboratories, Cancer Research UK, London Research Institute, Blanche Lane, South Mimms, Hertfordshire EN6 3LD, United Kingdom.

ABSTRACT
DNA helicases of the RECQ family are important for maintaining genome integrity, from bacteria to humans. Although progress has been made in understanding the biochemical role of some human RECQ helicases, that of RECQL5 remains elusive. We recently reported that RECQL5 interacts with RNA polymerase II (RNAPII), pointing to a role for the protein in transcription. Here, we show that RECQL5 inhibits both initiation and elongation in transcription assays reconstituted with highly purified general transcription factors and RNAPII. Such inhibition is not observed with the related, much more active RECQL1 helicase or with a version of RECQL5 that has normal helicase activity but is impaired in its ability to interact with RNAPII. Indeed, RECQL5 helicase activity is not required for inhibition. We discuss our findings in light of the fact that RECQ5(-/-) mice have elevated levels of DNA recombination and a higher incidence of cancer.

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Related in: MedlinePlus

RECQL5 inhibits an early step of RNAPII transcription. A, An outline of the experiment is shown. RECQL5 was either added during or after PIC assembly. B, Sarkosyl-treated RNAPII transcription reactions were treated with 0.9 pmol of the indicated RECQL5 protein, resolved by 6% denaturing PAGE, and visualized by phosphorimaging. The arrow indicates a 254-nt run-off transcript. Quantification of this transcript, relative to the control (lane 2, set to 1), is indicated below the lanes.
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Figure 4: RECQL5 inhibits an early step of RNAPII transcription. A, An outline of the experiment is shown. RECQL5 was either added during or after PIC assembly. B, Sarkosyl-treated RNAPII transcription reactions were treated with 0.9 pmol of the indicated RECQL5 protein, resolved by 6% denaturing PAGE, and visualized by phosphorimaging. The arrow indicates a 254-nt run-off transcript. Quantification of this transcript, relative to the control (lane 2, set to 1), is indicated below the lanes.

Mentions: RNAPII elongation complexes were prepared by performing in vitro transcription reactions with oligo(dC)-tailed DNA template derived from pAd-GR220. The final reaction contained 26 mm Tris-HCl, pH 7.9, 10.6 mm MgCl2, 1.3 mm DTT, 0.6 mg/ml bovine serum albumin, 10 units of RNasin (Promega), 133 ng of oligo(dC)-tailed DNA template, 0.6% polyvinyl alcohol, 17.7% (v/v) buffer D (20 mm HEPES-KOH, pH 7.9, 20% glycerol, 0.1 mm EDTA, and 0.1 m KCl), and ∼0.2 μg of rat liver RNAPII. U-less transcription was started by the addition of NTP mixture to give a final concentration of 50 μm ATP, 50 μm GTP, 2 μm CTP, and 10 μCi of [α-32P]CTP and incubated at 30 °C for 40 min. Stalled elongation complexes were chased to longer transcripts by the addition of UTP and CTP to a final concentration of 2 μm and 20 μm, respectively. Transcription elongation was performed at 30 °C for increasing amounts of time as described in the legend to Fig. 4 in the presence or absence of 0.9 pmol RECQ protein or after addition of ELL/EAF (as previously described (14)). Reactions were stopped, and RNA was purified and analyzed as described above.


Direct inhibition of RNA polymerase II transcription by RECQL5.

Aygün O, Xu X, Liu Y, Takahashi H, Kong SE, Conaway RC, Conaway JW, Svejstrup JQ - J. Biol. Chem. (2009)

RECQL5 inhibits an early step of RNAPII transcription. A, An outline of the experiment is shown. RECQL5 was either added during or after PIC assembly. B, Sarkosyl-treated RNAPII transcription reactions were treated with 0.9 pmol of the indicated RECQL5 protein, resolved by 6% denaturing PAGE, and visualized by phosphorimaging. The arrow indicates a 254-nt run-off transcript. Quantification of this transcript, relative to the control (lane 2, set to 1), is indicated below the lanes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2749093&req=5

Figure 4: RECQL5 inhibits an early step of RNAPII transcription. A, An outline of the experiment is shown. RECQL5 was either added during or after PIC assembly. B, Sarkosyl-treated RNAPII transcription reactions were treated with 0.9 pmol of the indicated RECQL5 protein, resolved by 6% denaturing PAGE, and visualized by phosphorimaging. The arrow indicates a 254-nt run-off transcript. Quantification of this transcript, relative to the control (lane 2, set to 1), is indicated below the lanes.
Mentions: RNAPII elongation complexes were prepared by performing in vitro transcription reactions with oligo(dC)-tailed DNA template derived from pAd-GR220. The final reaction contained 26 mm Tris-HCl, pH 7.9, 10.6 mm MgCl2, 1.3 mm DTT, 0.6 mg/ml bovine serum albumin, 10 units of RNasin (Promega), 133 ng of oligo(dC)-tailed DNA template, 0.6% polyvinyl alcohol, 17.7% (v/v) buffer D (20 mm HEPES-KOH, pH 7.9, 20% glycerol, 0.1 mm EDTA, and 0.1 m KCl), and ∼0.2 μg of rat liver RNAPII. U-less transcription was started by the addition of NTP mixture to give a final concentration of 50 μm ATP, 50 μm GTP, 2 μm CTP, and 10 μCi of [α-32P]CTP and incubated at 30 °C for 40 min. Stalled elongation complexes were chased to longer transcripts by the addition of UTP and CTP to a final concentration of 2 μm and 20 μm, respectively. Transcription elongation was performed at 30 °C for increasing amounts of time as described in the legend to Fig. 4 in the presence or absence of 0.9 pmol RECQ protein or after addition of ELL/EAF (as previously described (14)). Reactions were stopped, and RNA was purified and analyzed as described above.

Bottom Line: Here, we show that RECQL5 inhibits both initiation and elongation in transcription assays reconstituted with highly purified general transcription factors and RNAPII.Such inhibition is not observed with the related, much more active RECQL1 helicase or with a version of RECQL5 that has normal helicase activity but is impaired in its ability to interact with RNAPII.Indeed, RECQL5 helicase activity is not required for inhibition.

View Article: PubMed Central - PubMed

Affiliation: Mechanisms of Transcription Laboratory, Clare Hall Laboratories, Cancer Research UK, London Research Institute, Blanche Lane, South Mimms, Hertfordshire EN6 3LD, United Kingdom.

ABSTRACT
DNA helicases of the RECQ family are important for maintaining genome integrity, from bacteria to humans. Although progress has been made in understanding the biochemical role of some human RECQ helicases, that of RECQL5 remains elusive. We recently reported that RECQL5 interacts with RNA polymerase II (RNAPII), pointing to a role for the protein in transcription. Here, we show that RECQL5 inhibits both initiation and elongation in transcription assays reconstituted with highly purified general transcription factors and RNAPII. Such inhibition is not observed with the related, much more active RECQL1 helicase or with a version of RECQL5 that has normal helicase activity but is impaired in its ability to interact with RNAPII. Indeed, RECQL5 helicase activity is not required for inhibition. We discuss our findings in light of the fact that RECQ5(-/-) mice have elevated levels of DNA recombination and a higher incidence of cancer.

Show MeSH
Related in: MedlinePlus