Several lines of evidence indicate that convergent transcription through a CAG repeat tract triggers apoptosis via a DNA damage response pathway.27
First, induction of convergent transcription leads to activation of ATR (ATM and Rad3-related)—a major transducer protein kinase of the damage response—via phosphorylation at serine 428; ATR-dependent activation of cell cycle checkpoint kinase 1 (CHK1) via phosphorylation at serine 345; and ATR-dependent activation of p53 by phosphorylation at serine 15. The second major transducer kinase, ATM (ataxia-telangiectasia mutated), is activated with slower kinetics than ATR, via a pathway that does not depend on ATR; nor is ATM responsible for the observed phosphorylation of CHK1 and p53.27
Second, components of the ATR pathway, including ATR itself, ATRIP and TOPBP1, are recruited to the CAG repeat tract after induction of convergent transcription, as determined by ChIP analysis.27
Third, chemical and siRNA-mediated inactivation of components of the ATR pathway increases the fraction of the cell population that die when convergent transcription is induced.27
Thus, activation of the ATR pathway normally acts to suppress apoptosis, presumably by stimulating repair of the DNA structures that initiated the response.
The critical, ATR-activating DNA structures generated by convergent transcription through a CAG repeat tract are unknown, but they were not produced by sense or antisense transcription alone, nor did they occur in the absence of a CAG repeat. These considerations suggest that the problem may arise from the fusion of sense and antisense transcription bubbles, which we will refer to as a double bubble. illustrates several abnormal features of a double bubble that may be important for an ATR response.
Figure 2 Speculative model for induction of the ATR pathway by convergent transcription through a CAG repeat tract. RNAPII complexes are envisioned to stall at CAG and CTG hairpins on the separated template strand of the double bubble. The hairpins may be stabilized (more ...)
First, RNAPII complexes may stall at repeat-generated hairpins on both strands. ChIP analysis indicates that RNAPII accumulates at repeat tracts during convergent transcription.27
While it is known that CAG repeats can arrest RNAPII during transcription in vitro,44
CNG hairpins may be converted to more efficient RNAPII roadblocks by binding the mismatch repair (MMR) recognition complex, MSH2/3 (not depicted in ), as they do in vitro.45,46
In a similar way, O6
-methylguanine can be converted from a nonblocking lesion to one that blocks RNAPII by the binding of MMR proteins.47
The involvement of MSH2/3 in the formation of double bubbles fits with evidence that shows that they also promote transcription-induced CAG repeat instability in human cells.20,22
Second, the single-strand DNA binding protein, replication protein A (RPA) may coat single-strands of DNA on one or both sides of a double bubble (). ChIP analysis showed that RPA accumulates at CAG repeats when convergent transcription is induced.27
RPA might be expected to bind to single-stranded DNA adjacent to hairpins, contributing to the stability of the separated DNA strands. As discussed below, RPA-coated single strands are a critical element of the classic pathway for activating the ATR response.
Finally, RNA-DNA (R-loops) hybrids might be critical to the formation of an ATR-inducing DNA structure (). Studies in bacteria and mammalian cells demonstrated that R-loops form at CAG repeats during sense transcription.48,49
Stable R-loops are thought to form in CNG repeats due to the high thermal stability of rG/dC and rC/dG nucleotide pairs relative to dG/dC pairs.50,51
A recent study using in vitro transcription demonstrated that R-loops can form on DNA strands during convergent transcription through CNG repeat tracts.52
R-loops contribute to repeat instability since their persistence, due to genetic or siRNA mediated deficiency of RNase H, stimulates CAG repeat instability in both bacteria and human cells.48
These proposed features of convergent transcription-induced double bubbles do not link directly to the classic signal for ATR activation, which is RPA-coated single stranded tail protruding from a segment of double-stranded DNA.53
RPA-ssDNA localizes ATR and its binding partner ATRIP to the DNA, while the Rad9-Rad1-Hus1 (9-1-1) complex binds to the adjacent double stranded DNA. Interaction between the ATR-ATRIP and the 9-1-1 complexes allows binding of TOPBP1 (topoisomerase II binding protein 1), which contains the activation domain required for triggering the ATR signaling pathway.
What is missing from the double bubble in is the dsDNA-ssDNA junction. A nick adjacent to a hairpin could create such a junction, but it is unknown whether a hairpin, with its mismatches, could serve at the dsDNA component of the ATR signal. Alternatively, a nick could allow reannealing of adjacent ssDNA to create a suitable dsDNA segment with ssDNA tail. A natural candidate for introducing nicks would be nucleotide excision repair (NER), which is known to be involved in repeat instability induced by transcription through a CAG repeat.23,54
In the absence of its usual dsDNA substrate, NER might operate abortively to nick a double bubble, allowing formation of the classic structure. Finally, it is conceivable that the end of an RNA-DNA hybrid (if RNPII were removed) could serve as the necessary double-stranded junction, with the template DNA strand forming the ssDNA tail.
It is also possible that the features of the double bubble in may be adequate by themselves, since some reports suggest that ATR activation may not be solely dependent on the classic structure.53
For example, constitutive nuclear translocation of the ATR-activation domain of TOPBP1 is enough to activate the ATR pathway, including phosphorylation of CHK1 and p53, in the absence of any obvious DNA damage.55
In addition, in a system that reconstituted the human ATR-mediated checkpoint response to bulky lesions, purified components were used to show that ATR-ATRIP phosphorylates CHK1 in a reaction that requires TOPBP1, is strongly dependent on DNA containing bulky base lesions, but appears to be entirely independent of DNA ends.56,57
Finally, if the classic structure were the only way to activate ATR, it might be expected that the phenotypes generated by the loss of ATR or by the loss of the 9-1-1 complex would be the same; however, the loss of ATR is much more severe than the loss of RAD9 or HUS1.53
Although neither mechanism has been defined, convergent transcription-induced ATR activation resembles the transcriptional stress response, which also involves the ATR- and RPA-dependent activation of CHK1 and p53.58–60
Interference with the progression of the RNAPII complex by treatments such as UV light, actinomycin D and psoralen (which cause pyrimidine dimers, base intercalation and interstrand crosslinks, respectively) activate the ATR pathway.60
But DNA damage is not required since antibodies to the elongating form of RNAPII elicit the same ATR response.60
Thus, it may be that a stalled RNAPII complex in the presence of RPA-coated ssDNA is sufficient to stimulate an ATR response; that is, that the stalled RNAPII complex may serve directly as the sensor for transcriptional stress and triggers the cellular response.54,61
What is remarkable about convergent transcription through a CAG repeat tract is that a single genomic site of transcriptional interference—a single toxic site—is capable of triggering an ATR response that can lead to cell death.