In trying to define a fundamental role for BRCA1 in breast cancer, we took advantage of the evolutionary distance between humans and yeast to identify proteins and/or processes that have the ability to interact functionally with BRCA1 in yeast; these BRCA1-interacting partners were identified by finding yeast deletions that suppress BRCA1-induced lethality. For such “targets” to be biologically relevant, the yeast genes and human orthologs would most likely play roles in DNA damage responses and genome stability, and they would have an increased likelihood to physically interact with the BRCA1 protein. We present evidence that fulfills these criteria for proteins in the DSIF complex (SPT4/SPT5). Further, DSIF and other conserved suppressors of BRCA1-induced lethality may be grouped into a temporally linked mRNA signaling/decay pathway that responds to DNA damage encountered by RNAPII in actively transcribing genes at the elongation stage of transcription (). Specifically, we identified BRCA1 suppressors that participate in mRNA transcription elongation as well as mRNA export and decay. Some of the suppressors (CCR4
) were first identified and characterized as IR resistance genes in a RAD9
-dependent, checkpoint pathway required for G1/S transition following DNA damage 
. This checkpoint role for CCR4
has been subsequently confirmed by other laboratories    
and a role for BRCA1 in G1/S checkpoint arrest following IR damage has been previously described 
. Similarly, following overexpression of BRCA1 in human cells, a G1 arrest was observed 
The two yeast genes whose deletion most potently suppressed BRCA1-induced G1 arrest and lethality were those of the conserved transcription elongation factor Spt4p and the elongation phase-specific CTD kinase I (Ctk1-kinase), underlying the critical role that transcription elongation and RNAPII CTD phosphorylation play in BRCA1-mediated lethality. Furthermore, in agreement with previously described physical interactions observed between BRCA1 and human RNAPII  
, BRCA1 was found to interact with the P-CTD of RNAPII in yeast resulting in P-CTD cleavage. It should be noted that the ethanol procedure used to prevent BRCA1 degradation extracts a relatively small fraction of the total cellular protein. Therefore, the amount of RNAPII that undergoes cleavage is unclear at this time.
Disease-associated mutations in BRCA1 that are expressed in this system neither reduce the viability of the yeast nor result in P-CTD cleavage suggesting that these events may be causally linked. Further support for the association of P-CTD cleavage and lethality comes from the rescue of both phenotypes in the def1Δ
strain, previously shown to be defective for ubiquitin-mediated RNAPII degradation following DNA damage 
. These results implicate BRCA1 in DNA damage-mediated degradation of elongating RNAPII via cleavage of the P-CTD. The highly conserved nature of the Rpb1p CTD suggested that similar interactions occur in human cells. Our results in human breast cancer cell lines fully support this interpretation. We have shown that P-CTD fragments exist in breast cancer cell lines, are stabilized by proteasome inhibition, and depend upon the presence of wild type BRCA1. Reconstitution of the damage-induced P-CTD accumulation following ectopic expression of wild type BRCA1 in the mutant HCC1937 cell line provides direct evidence for this process.
Results from ectopic expression in yeast of mutant BRCA1 proteins and from examination of human HCC1937 cells (expressing a carboxy terminal BRCA1 deletion) both point to the BRCT domain of BRCA1 as the mediator of P-CTD cleavage and associated lethality in yeast. Although the BRCA1-BRCT domain is necessary to promote P-CTD cleavage, it is not sufficient. For example, the BRCA1(ΔM) construct (that contains intact RING and BRCT domains) fails to reconstitute BRCA1-induced P-CTD cleavage following DNA damage. Furthermore, the BRCA1-ΔM construct can promote ubiquitination of intact RPB1 
, suggesting that P-CTD cleavage and ubiquitination are separate processes. Moreover, the relative ease in transiently overexpressing the BRCA1-ΔM construct 
compared to the wild type may be related to this defect in P-CTD cleavage.
A critical role for BRCA1 and its stochiometric binding partner BARD1 in ubiquitin-mediated degradation of phosphorylated RNAPII has been previously described in vitro  
. Deletion of DEF1
inhibits BRCA1-mediated RNAPII CTD cleavage and degradation much in the same manner as ubiquitin targeted RNAPII degradation is inhibited following UV in def1Δ
. This suggests that BRCA1 subverts a conserved RNAPII degradation response to DNA damage to induce lethality in yeast. Following DNA damage, Def1p may be essential to couple arrested RNAPII to the proteasome to facilitate its degradation 
BRCA1 associates preferentially with the hyperphosphorylated form of RNAPII   
and appears to modulate RNAPII CTD phosphorylation levels 
. Our finding that loss of the CTD specific kinase gene CTK1
completely suppresses BRCA1-induced lethality indicates that BRCA1 interacts preferentially with the phosphorylated elongating form of the RNAPII specified by CTDK-I (i.e
. phosphorylated on Ser2 and Ser5). The preferential interaction of BRCT domains with phosphoserine 
suggests a potential physical interaction between BRCA1 and the P-CTD and predicts a critical role for the conserved human Ctk1p ortholog CRKRS (see CTK1
in Table S1
). Others have reported that, upon treatment with DNA damaging agents, the association of BRCA1 with RNAPII was disrupted suggesting a link between DNA damage signaling/repair and transcription 
. The apparent damage-induced disruption of BRCA1 interaction with RNAPII may reflect degradation of RNAPII following DNA damage, dependent on specific co-factors such as DSIF as suggested in this study. The finding of enhanced phosphorylation and subsequent proteasome-mediated degradation of RNAPII that involves P-CTD cleavage following DNA damage in both yeast ( 
(this study) and human cells  
(this study) further illustrates the important conserved role these ubiquitin-mediated degradation processes play in the BRCA1 damage response. Moreover, the finding that proteasome mediated degradation of BRCA1 occurs primarily in G1 
supports a spontaneous role for BRCA1 in degrading RNAPII in the absence of damage. Alternatively, spontaneous damage within transcriptionally active genes may account for the P-CTD cleavage observed in unirradiated breast epithelial cells ().
Interaction of BRCA1 with the negative elongation factor NELF-B/COBRA1 
combined with the finding that COBRA1, which, as part of the NELF complex interacts with DSIF to negatively regulate RNAPII transcription 
is fully supportive of our determination that the DSIF complex also interacts genetically and physically with BRCA1 and suggests a damage signaling role for both COBRA1 and DSIF in concert with BRCA1 and RNAPII. Our study supports () an emerging model that human cells employ the RNAPII holoenzyme complex during transcription elongation for DNA damage surveillance  
. In this respect, BRCA1 may act as a sensor of DNA damage to monitor stalling of the transcription apparatus at DNA lesions and, therefore, participates in a transcriptional checkpoint to signal the presence of lesions within actively transcribing genes 
. As suggested by this study, a G1 checkpoint arrest signal might be maintained until subsequent transport of the prematurely terminated transcript for degradation at P-bodies and disassembly of the RNAPII-BRCA1-DSIF complex by ubiquitin-mediated degradation at the proteasome (). Our finding that the human ortholog of Dhh1p (DDX6) interacts with BRCA1 in the cytoplasm at P-bodies following DNA damage also supports such a model (T. Westmoreland and J. Marks, unpublished). The determination that Dhh1p (and its human ortholog, DDX6) is an essential component of processing bodies that are sites of mRNA decay  
allows us to functionally link transcription elongation arrest by DNA damage to mRNA export and decay through BRCA1 and P-CTD cleavage (). Thus BRCA1 may facilitate repair of DNA damage by mediating P-CTD cleavage and subsequent degradation of stalled transcription complexes to allow enhanced access of repair complexes to the lesion site. This model also accommodates reports that BRCA1 participates in transcription-coupled repair  
and is supported by the finding that overexpression of hTREX84 and dysregulation of mRNA export may be a hallmark of breast tumors 
. Taken together our data support BRCA1 playing a critical role in a novel checkpoint pathway that senses DNA damage within actively transcribed DNA to initiate cleavage and degradation of stalled RNAPII elongation complexes. Furthermore, our results showing BRCA1-BRCT function is required for the rapid alkylation-induced degradation of BRCA1-SPT5 and RNAPII suggests that chemotherapeutic treatments utilizing alkylating agents may be clinically effective for treating breast cancers in which BRCA1-mediated degradation is defective. Moreover, the use of proteasome inhibitors in concert with alkylating agents may be equally effective for treating those breast cancers in which the damage-induced BRCA1-SPT5-RNAPII degradation pathway remains intact. Defects in this conserved BRCA1-dependent RNAPII cleavage and degradation pathway may be critical for the initiation of breast or ovarian cancer, predicting that genetic defects in other conserved components of this pathway may also contribute to these diseases.