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Direct inhibition of RNA polymerase II transcription by RECQL5.

AygΓΌn 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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RECQL5 inhibits reconstituted RNAPII transcription. A, outline of the experiment. PIC, pre-initiation complex. B, 0.113, 0.225, and 0.9 pmol of RECQL5 (lanes 2–4) or RECQL5ID (lanes 5–7) were added during preinitiation complex formation. Transcripts were resolved by 6% denaturing PAGE and visualized by phosphorimaging. The arrow indicates a 254-nt run-off transcript. C, effect of RECQL5D157A and RECQL1 as described in B. The experiments in B and C were performed at different times and thus cannot be used for precise quantitative comparisons of the relative effects of RECQL5 and RECQL5D157A. Please note that the experiments in Figs. 5 and 6 show that RECQL5D157A does not inhibit transcription more potently than wild type RECQL5.
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Figure 3: RECQL5 inhibits reconstituted RNAPII transcription. A, outline of the experiment. PIC, pre-initiation complex. B, 0.113, 0.225, and 0.9 pmol of RECQL5 (lanes 2–4) or RECQL5ID (lanes 5–7) were added during preinitiation complex formation. Transcripts were resolved by 6% denaturing PAGE and visualized by phosphorimaging. The arrow indicates a 254-nt run-off transcript. C, effect of RECQL5D157A and RECQL1 as described in B. The experiments in B and C were performed at different times and thus cannot be used for precise quantitative comparisons of the relative effects of RECQL5 and RECQL5D157A. Please note that the experiments in Figs. 5 and 6 show that RECQL5D157A does not inhibit transcription more potently than wild type RECQL5.

Mentions: To study the potential effect of RECQL5 on transcription, we reconstituted mammalian RNAPII transcription using highly purified general transcription factors (GTFs), RNAPII, and a DNA template containing the adenovirus major late promoter (16). Then, the effect of increasing amounts of purified RECQ proteins on production of the run-off transcript was analyzed (Fig. 3A). RECQL5 protein was inhibitory for basal RNAPII transcription, and inhibition increased as larger amounts of the protein was added (Fig. 3B, compare lanes 1–4). The RECQL5ID protein had little or no effect (Fig. 3B, compare lanes 2–4 and 5–7), indicating that inhibition was not merely due to the addition of a DNA-unwinding helicase activity but required a stable RECQL5-RNAPII interaction. Indeed, the much more active RECQL1 helicase failed to affect transcription, further indicating that the effect observed with RECQL5 was specific (Fig. 3C, lanes 5–7). Finally, the RECQL5D157A helicase mutant also inhibited basal transcription (Fig. 3C, lanes 2–4), indicating that RECQL5-mediated inhibition of RNAPII basal transcription does not require its helicase activity.


Direct inhibition of RNA polymerase II transcription by RECQL5.

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

RECQL5 inhibits reconstituted RNAPII transcription. A, outline of the experiment. PIC, pre-initiation complex. B, 0.113, 0.225, and 0.9 pmol of RECQL5 (lanes 2–4) or RECQL5ID (lanes 5–7) were added during preinitiation complex formation. Transcripts were resolved by 6% denaturing PAGE and visualized by phosphorimaging. The arrow indicates a 254-nt run-off transcript. C, effect of RECQL5D157A and RECQL1 as described in B. The experiments in B and C were performed at different times and thus cannot be used for precise quantitative comparisons of the relative effects of RECQL5 and RECQL5D157A. Please note that the experiments in Figs. 5 and 6 show that RECQL5D157A does not inhibit transcription more potently than wild type RECQL5.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: RECQL5 inhibits reconstituted RNAPII transcription. A, outline of the experiment. PIC, pre-initiation complex. B, 0.113, 0.225, and 0.9 pmol of RECQL5 (lanes 2–4) or RECQL5ID (lanes 5–7) were added during preinitiation complex formation. Transcripts were resolved by 6% denaturing PAGE and visualized by phosphorimaging. The arrow indicates a 254-nt run-off transcript. C, effect of RECQL5D157A and RECQL1 as described in B. The experiments in B and C were performed at different times and thus cannot be used for precise quantitative comparisons of the relative effects of RECQL5 and RECQL5D157A. Please note that the experiments in Figs. 5 and 6 show that RECQL5D157A does not inhibit transcription more potently than wild type RECQL5.
Mentions: To study the potential effect of RECQL5 on transcription, we reconstituted mammalian RNAPII transcription using highly purified general transcription factors (GTFs), RNAPII, and a DNA template containing the adenovirus major late promoter (16). Then, the effect of increasing amounts of purified RECQ proteins on production of the run-off transcript was analyzed (Fig. 3A). RECQL5 protein was inhibitory for basal RNAPII transcription, and inhibition increased as larger amounts of the protein was added (Fig. 3B, compare lanes 1–4). The RECQL5ID protein had little or no effect (Fig. 3B, compare lanes 2–4 and 5–7), indicating that inhibition was not merely due to the addition of a DNA-unwinding helicase activity but required a stable RECQL5-RNAPII interaction. Indeed, the much more active RECQL1 helicase failed to affect transcription, further indicating that the effect observed with RECQL5 was specific (Fig. 3C, lanes 5–7). Finally, the RECQL5D157A helicase mutant also inhibited basal transcription (Fig. 3C, lanes 2–4), indicating that RECQL5-mediated inhibition of RNAPII basal transcription does not require its helicase activity.

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