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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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Activity of the recombinant RECQ proteins used in this study. A, Coomassie-stained SDS-PAGE gel showing recombinant RECQ proteins. M, protein molecular weight standard marker. B, DNA helicase assays showing the relative ATP-dependent helicase activities of the recombinant proteins shown in A.
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Figure 2: Activity of the recombinant RECQ proteins used in this study. A, Coomassie-stained SDS-PAGE gel showing recombinant RECQ proteins. M, protein molecular weight standard marker. B, DNA helicase assays showing the relative ATP-dependent helicase activities of the recombinant proteins shown in A.

Mentions: Next, we expressed wild type RECQL5, the RNAPII interaction-deficient protein (RECQL5ID), a RECQL5 helicase-deficient point mutant (RECQL5D157A), and another human RECQ family helicase, RECQL1, in E. coli, and purified the recombinant proteins to virtual homogeneity (Fig. 2A). These proteins were then tested in ATP-dependent helicase assays (Fig. 2B). As expected, the RECQL5D157A mutant did not exhibit helicase activity (Fig. 2B, lanes 4–5) but was fully capable of interacting with RNAPII (data not shown). RECQL1 exhibited robust helicase activity, much greater than that of RECQL5 (Fig. 2B, compare lanes 8–9 with 2–3). Most importantly, RECQL5 and RECQL5ID had similar activity (Fig. 2B, compare lanes 2–3 and 6–7), indicating that the internal deletion in RECQL5ID (which greatly reduced RNAPII interaction) does not significantly affect overall protein folding and enzymatic activity.


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)

Activity of the recombinant RECQ proteins used in this study. A, Coomassie-stained SDS-PAGE gel showing recombinant RECQ proteins. M, protein molecular weight standard marker. B, DNA helicase assays showing the relative ATP-dependent helicase activities of the recombinant proteins shown in A.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Activity of the recombinant RECQ proteins used in this study. A, Coomassie-stained SDS-PAGE gel showing recombinant RECQ proteins. M, protein molecular weight standard marker. B, DNA helicase assays showing the relative ATP-dependent helicase activities of the recombinant proteins shown in A.
Mentions: Next, we expressed wild type RECQL5, the RNAPII interaction-deficient protein (RECQL5ID), a RECQL5 helicase-deficient point mutant (RECQL5D157A), and another human RECQ family helicase, RECQL1, in E. coli, and purified the recombinant proteins to virtual homogeneity (Fig. 2A). These proteins were then tested in ATP-dependent helicase assays (Fig. 2B). As expected, the RECQL5D157A mutant did not exhibit helicase activity (Fig. 2B, lanes 4–5) but was fully capable of interacting with RNAPII (data not shown). RECQL1 exhibited robust helicase activity, much greater than that of RECQL5 (Fig. 2B, compare lanes 8–9 with 2–3). Most importantly, RECQL5 and RECQL5ID had similar activity (Fig. 2B, compare lanes 2–3 and 6–7), indicating that the internal deletion in RECQL5ID (which greatly reduced RNAPII interaction) does not significantly affect overall protein folding and enzymatic 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