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. 1997 Oct 14;94(21):11205-9.
doi: 10.1073/pnas.94.21.11205.

Cockayne syndrome group B protein enhances elongation by RNA polymerase II

C P Selby  1 , A Sancar
Affiliations

Cockayne syndrome group B protein enhances elongation by RNA polymerase II

C P Selby et al. Proc Natl Acad Sci U S A. .

Abstract

Cockayne syndrome (CS) is characterized by impaired physical and mental development. Two complementation groups, CSA and CSB, have been identified. Here we report that the CSB gene product enhances elongation by RNA polymerase II. CSB stimulated the rate of elongation on an undamaged template by a factor of about 3. A thymine-thymine cyclobutane dimer located in the template strand is known to be a strong block to transcription. Addition of CSB to the blocked polymerase resulted in addition of one nucleotide to the nascent transcript. Finally, addition of transcription factor IIS is known to cause polymerase blocked at a thymine-thymine cyclobutane dimer to digest its nascent transcript, and CSB counteracted this transcript shortening action of transcription factor IIS. Thus a deficiency in transcription elongation may contribute to the CS phenotype.

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Figures

Figure 1
Figure 1
CSB stimulates transcription elongation. RNAPII was stalled at the end of the 112-nucleotide long U-less cassette in pMLU112 by omission of UTP. (A) CSB was added to 9 nM (lanes 3 and 5), then reactions were chased by adding UTP, and CTP was added to dilute the radiolabel. After 0–3 min of chasing, reactions were stopped and resolved on a sequencing gel alongside DNA markers (lane M) of the sizes indicated. The effect of CSB was optimal when it was present at 6–20 nM. (B) The CSB preparation was immunoprecipitated in the presence and absence of anti-CSB antibody. Gel analysis (not shown) indicated that the antibody did in fact precipitate CSB protein. The supernatants were added to transcription reactions with CSB nominally at 10 nM, and transcription was chased as in A for 1 min
Figure 2
Figure 2
CSB-RNAPII interaction. A pull-down assay utilized MBP-CSB, a fusion protein of amino acids 528-1222 of CSB fused to the C terminus of MBP (13). MBP and the MBP-CSB fusion protein, “MBPE65” each attached to amylose at 1 g/l, were incubated with purified RNAPII. Resins were pelleted and unbound (Free) proteins in the supernatants were removed. Resins with “Bound” proteins were washed. Components of Free and Bound fractions were separated on a SDS/polyacrylamide gel and analyzed by Western blot using monoclonal anti-RNAPII C-terminal domain antibodies (Promega). Both phosphorylated (RNAPIIo) and nonphosphorylated (RNAPIIa) forms of RNAPII bound to CSB.
Figure 3
Figure 3
Transcription termination site in relation to the T<>T. In Panel A, end labeled transcripts terminated at the T<>T were digested 3′ to G residues using 1/100,000 (lane 2) and 1/30,000 (lane 3) dilutions of RNase T1 (1931 units/μl, GIBCO/BRL). Products were resolved alongside undigested RNA (lane 1) and RNA ladders (L) generated by alkaline hydrolysis. Lines to the right indicate RNA residues. G residues and termination sites a, b, c, and P are labeled. (B) Transcripts made from unmodified pPU192, pPU192 digested with XhoI, or pPU192 with a T<>T in the template strand. (C) DNA and RNA sequences surrounding the T<>T (indicated with a bracket). Termination sites a, b, c, and P and three major cleavage products obtained in the presence of TFIIS are labeled above the RNA. Arrows below indicate where the excision repair nuclease nicks the damaged strand 3′ to the lesion (25). The RNA sequence opposite the dimer may vary from that shown due to the damage.
Figure 4
Figure 4
Effects of CSB plus TFIIS on blocked RNAPII. (A) Stalled elongation complexes were formed at the T<>T located in the template strand of pPU192. Reactions were then incubated with CSB and TFIIS for 25 min at 30°C. Transcripts a, b, c, and P are indicated. The experiment shown in B utilized RNAPII stalled at the end of the 112-nucleotide U-less cassette in pMLU112. (B) CSB and TFIIS were incubated with the stalled complex for 25 min at 30°C.
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References

    1. Nance M A, Berry S A. Am J Med Genet. 1992;42:68–84. - PubMed
    1. Friedberg E C, Walker G C, Siede W. DNA Repair Mechanisms. Washington, DC: Am. Soc. Microbiol.; 1995.
    1. Mayne L V, Lehmann A R. Cancer Res. 1982;42:1473–1478. - PubMed
    1. Venema J, Mullenders L H, Natarajan A T, van Zeeland A A, Mayne L V. Proc Natl Acad Sci USA. 1990;87:4707–4711. - PMC - PubMed
    1. Bohr V A, Smith C A, Okumoto D S, Hanawalt P C. Cell. 1985;40:359–369. - PubMed

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