Structural Characterization of RNA Polymerase II Complexes Arrested by a Cyclobutane Pyrimidine Dimer in the Transcribed Strand of Template DNA

Abstract

We have characterized the properties of immunopurified transcription complexes arrested at a specifically located cyclobutane pyrimidine dimer (CPD) using enzymatic probes and an in vitro transcription system with purified RNA polymerase II (RNAP II) and initiation factors. To help understand how RNAP II distinguishes between a natural impediment and a lesion in the DNA to initiate a repair event, we have compared the conformation of RNAP II complexes arrested at a CPD with complexes arrested at a naturally occurring elongation impediment. The footprint of RNAP II arrested at a CPD, using exonuclease III and T4 DNA polymerase's 335 exonuclease, covers ϳ35 base pairs and is asymmetrically located around the dimer. A similar footprint is observed when RNAP II is arrested at the human histone H3.3 arrest site. Addition of elongation factor SII to RNAP II arrested at a CPD produced shortened transcripts of discrete lengths up to 25 nucleotides shorter than those seen without SII. After addition of photolyase and exposure to visible light, some of the transcripts could be reelongated beyond the dimer, suggesting that SII-mediated transcript cleavage accompanied significant RNAP II backup, thereby providing access of the repair enzyme to the arresting CPD. Transcription-coupled repair (TCR)1 is a subpathway of nucleotide excision repair that removes lesions from the transcribed strand of actively transcribed genes (1). TCR has been shown to occur in mammalian cells (2), in Escherichia coli (3), and in Saccharomyces cerevisiae (4 -6). Several lines of evidence have suggested that an active RNA polymerase elongation complex is necessary for preferential repair of the transcribed strand (7). A model for TCR has been proposed in which the RNA polymerase stalled at a lesion directs repair enzymes to the transcribed strand of an active gene (2). This model assumes that the polymerase must be removed from the site of the lesion to provide access for the repair complex to the lesion site and to allow reannealing of the DNA strands to form a proper substrate for repair.An essential question about the mechanism of TCR is how the repair proteins recognize an RNAP II elongation complex arrested at a lesion and distinguish it from a transcription complex arrested at a natural arrest site. The process of transcriptional arrest can provide some clues to this question. RNAP II arrest sites have been identified and characterized (8).Arrest can occur at a bend in the helix axis of template DNA (9). It can also be induced by nucleotide depletion (10), DNA-binding drugs (11), and sequence-specific DNA-binding proteins (12). RNAP II can bypass arrest sites by activation of a cryptic endonuclease function that resides in the polymerase, a process mediated by the transcription elongation factor SII. SII-mediated transcript cleavage removes short oligonucleotides of discrete lengths from the 3Ј end of the nascent RNA. Transcript shortening is thought to restore the association of the 3Ј end of the transcript with the catalytic site in the po...

Photorepair of RNA polymerase arrest and apoptosis after ultraviolet irradiation in normal and XPB deficient rodent cells

“…Although, these lesions are normally shielded by the arrested polymerase complex, 11 under certain conditions this may be displaced, allowing for photolyase access to CPD and, thus, RNA elongation in vitro. 21 In this work, photoreactivation in CHOphr cells efficiently restore RNA synthesis in UV-irradiated cells. Although this restoration decreases with time after UV (e.g.…”

Malondialdehyde adducts in DNA arrest transcription by T7 RNA polymerase and mammalian RNA polymerase II

“…Most forms of damage targeted for repair by TCR also impede the translocation of RNAP complexes along DNA templates in vitro (6,8,33,35,36). Because bacteria, yeast, and mammals possess TCR, this mechanism may be evolutionarily conserved for efficient repair of DNA modifications that threaten genomic stability.…”

“…2). The constructs were transformed into the FЈ E. coli strain MV1184 for replication of the plus strand by the M13K07 phage (33). Plasmids containing dG, M 1 dG, or PdG in either the transcribed or nontranscribed strand were prepared by T4 DNA polymerase second-strand synthesis with the appropriate 18-mer as a primer.…”

“…The arrested transcripts produced by RNAPII in the above reactions could be released from stable RNAPII complexes by detergent denaturation or from the dissociation of unstable complexes soon after RNAPII translocation is blocked by the adducts. The stability of RNAPII complexes arrested at sites of DNA damage has been monitored by their response to the elongation factor SII (8,33). SII activates a cryptic ribonuclease activity of RNAPII and promotes RNAPII read-through of pause sequences during transcription (34).…”

The role of Cockayne Syndrome group B (CSB) protein in base excision repair and aging

“…RNAPII complex must be displaced and/or degraded in order for efficient repair because it shields the DNA lesion and prevents accessibility of NER proteins (reviewed in Licht et al, 2003;Fousteri et al, 2008). Several models have been proposed regarding how CSB rescues RNAPII complexes that are stalled at DNA lesions (Selby et al, 1997;Tornaletti et al, 1999), but the details of this process remains unclear. It has been suggested that CSB is involved in initiation of TCR through recognition of blocked RNAPII and binding to this complex, followed by recruitment of other NER proteins to the damaged site.…”