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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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A central 9-amino acid sequence is essential for RNAPII-RECQL5 interaction in vivo. A, diagram describing the constructs used. B and C, Western blots of FLAG-tagged immunoprecipitations (IP) showing interactions between different forms of FLAG-tagged RECQL5 and RNAPII, using FLAG and RPB1 antibody, respectively.
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Figure 1: A central 9-amino acid sequence is essential for RNAPII-RECQL5 interaction in vivo. A, diagram describing the constructs used. B and C, Western blots of FLAG-tagged immunoprecipitations (IP) showing interactions between different forms of FLAG-tagged RECQL5 and RNAPII, using FLAG and RPB1 antibody, respectively.

Mentions: To facilitate a study of the functional consequences of the RECQL5-RNAPII interaction, we first delineated a region in the RECQL5 protein that is required for normal association with the polymerase. We previously reported that the RECQL5-RNAPII interaction requires the C-terminal half of RECQL5 (12). To more precisely map domains that contribute to this interaction, we established stable human 293T cell lines expressing similar amounts of different FLAG-tagged forms of RECQL5 (Fig. 1, A–C). The RECQL5 protein was immunoprecipitated from the individual cell lines using anti-FLAG antibody, and the association with RNAPII was analyzed by anti-RPB1 Western blotting (Fig. 1, B and C, RPB1). As reported previously (12), full-length RECQL5 protein bound RNAPII (Fig. 1B, lane 2), whereas the RECQL5 (1–542) truncation mutant did not (lane 3). The remaining C-terminal part of the protein (541–991) was unable to bind RNAPII as well (lane 4), which could suggest that residues around amino acids 542 are critical for the interaction. Indeed, we discovered that an internal deletion of nine amino acids, located between positions 542 and 552 (RECQL5ID), dramatically decreased the interaction with RNAPII (Fig. 1C, compare lanes 1 and 2).


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)

A central 9-amino acid sequence is essential for RNAPII-RECQL5 interaction in vivo. A, diagram describing the constructs used. B and C, Western blots of FLAG-tagged immunoprecipitations (IP) showing interactions between different forms of FLAG-tagged RECQL5 and RNAPII, using FLAG and RPB1 antibody, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: A central 9-amino acid sequence is essential for RNAPII-RECQL5 interaction in vivo. A, diagram describing the constructs used. B and C, Western blots of FLAG-tagged immunoprecipitations (IP) showing interactions between different forms of FLAG-tagged RECQL5 and RNAPII, using FLAG and RPB1 antibody, respectively.
Mentions: To facilitate a study of the functional consequences of the RECQL5-RNAPII interaction, we first delineated a region in the RECQL5 protein that is required for normal association with the polymerase. We previously reported that the RECQL5-RNAPII interaction requires the C-terminal half of RECQL5 (12). To more precisely map domains that contribute to this interaction, we established stable human 293T cell lines expressing similar amounts of different FLAG-tagged forms of RECQL5 (Fig. 1, A–C). The RECQL5 protein was immunoprecipitated from the individual cell lines using anti-FLAG antibody, and the association with RNAPII was analyzed by anti-RPB1 Western blotting (Fig. 1, B and C, RPB1). As reported previously (12), full-length RECQL5 protein bound RNAPII (Fig. 1B, lane 2), whereas the RECQL5 (1–542) truncation mutant did not (lane 3). The remaining C-terminal part of the protein (541–991) was unable to bind RNAPII as well (lane 4), which could suggest that residues around amino acids 542 are critical for the interaction. Indeed, we discovered that an internal deletion of nine amino acids, located between positions 542 and 552 (RECQL5ID), dramatically decreased the interaction with RNAPII (Fig. 1C, compare lanes 1 and 2).

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