The mechanism by which hairpin N-methylpyrrole-imidazole polyamides (PAs) act to destabilize and eliminate HPV episomes from cells was examined. Evidence from PCR arrays, a siRNA screen, and functional studies suggests that DDR repair pathways mediate the antiviral activity of PA25. The MRN and the Rad9-Hus1-Rad1 (9-1-1) complexes, in particular, appear to play an important role in the massive HPV episome loss promoted by PA25. The expression of Rad1, Mre11 and Nbs1 genes was altered in response to PA25 treatment. Mre11, Rad1, and Rad9 also emerged in the siRNA screen as highly significant regulators of episome stability in the presence of PA25. These data, as well as Southern blots and functional studies, suggest that HPV DNA episome alteration or damage by PA25 is a key, rate-limiting feature of its antiviral activity. PAs bind to DNA and cause a number of biophysical effects such as widening the minor groove, shrinking the major groove, and stiffening the double helix 
. In the context of the HPV open circle or negatively super-coiled episome, PAs may cause twisting, exposure of ssDNA, or DSBs. In addition, various DNA repair pathways will recognize the bulky PAs bound to the minor groove as problematic. A broad DDR is mounted resulting in the elimination of episomes by a process that is poorly understood. It is important to note that, unlike the DDR elicited by damage to cellular genomes, the DDR elicited by PA25 does not result in robust cell cycle arrest or have large effects upon cell growth or apoptosis. We believe that this difference is attributable to both the unique niche occupied by HPV genomes within the nucleus and to the well-known action of the HPV oncogenes.
The damage to HPV episomes by PA25 is specific according to the following criteria: 1.
Inactive PAs do not trigger a DDR, 2.
PA25 activates a DDR response in HPV episome-bearing W12E cells that is qualitatively and quantitatively distinct from HPV-negative C33A cells and SiHa cells, which carry integrated copies of HPV16. These findings indicate that PA25 specifically acts on HPV in the context of the viral episome, 3.
PA25 triggers phosphorylation of Rad9 in HPV-positive W12E cells but not in C33A cells, and 4.
Pharmacological and siRNA inhibition of Mre11 potentiates PA25 antiviral activity by sensitizing HPV genomes. PA25 and other antiviral PAs do not cause measurable toxicity in HPV episome-bearing cells or HPV-negative cells indicating that these compounds do not significantly compromise cell genomes 
. These data are fully consistent with numerous reports in the literature attesting to the low toxicity of this class of compound 
It was possible to observe transient alterations in episome structure attributable to PA25 by focusing on the earliest times of treatment (). A retardation of migration of supercoiled DNA is noted at the earliest times after treatment. These changes in electrophoretic migration are attributed to structural alterations in the supercoiled episome, and not increased mass due to PA25 binding, since it is highly unlikely that PA25 survives the extensive deprotonation during the DNA isolation procedure. Also, no such alterations in electrophoretic migration occur in the matched, BamH1-linearized samples, indicating that an alternative, perhaps relaxed, structure of the HPV supercoil is responsible. In the presence of 10 µM PA25, the appearance of a series of HPV topoisomers over time was clearly observed (). We hypothesize that these topoisomers arise by topoisomerase resolution of PA25-generated stress within HPV supercoiled DNA. After 5 hours in the presence of 1 µM PA25, a band co-migrating with the 8 kb linearized HPV standard also appeared suggesting that dsDNA breaks are occurring within the altered supercoiled episome (). The data from end labeling of HPV genomes coupled to Q-PCR (ELCQ; ) also demonstrates the generation of PA25 induced breaks within the viral genomes.
It is important to note that PAs and other minor groove-binding agents affect DNA structure in a number of quantitative and qualitative ways 
. Most recently, 6- and 8-ring polyamides considerably smaller than PA25 were shown to affect DNA bending in a manner dependent on DNA and polyamide (PA) sequence. The 6-ring PA bent DNA 5.4° per helical turn while 8-ring PAs bent DNA 0, 3.0 and 3.7° per turn under conditions benchmarked by straight DNA and an A5
sequence bent 18° per turn 
. The stiffening of entire helical turns of DNA by PA25 could cause, for example, unequal distribution of supercoiling in an episome, resulting in unusual topologies or stretches of ssDNA that are recognized as damaged sites by the DDR. The work described here clearly shows that PA25 elicits a robust and complex DDR in cells carrying HPV episomes, but not in cells that have integrated HPV16 DNA (SiHa) or are HPV negative (C33A). DNA twisting and breaks may arise because of stress from PA25 binding within the HPV genome that is impossible to mitigate by unwinding because of its circular nature. Mre11 protects the viral genome from PA25 under these conditions.
The MRN complex is particularly interesting as a PA25 repressor. Both MRE11 and NBS1 genes are down-regulated in response to PA25 treatment, as is the MRN structural and functional partner CtIP (RBBP8), which mediates DSB resection during repair 
. MRE11 also emerged as a strong repressor of PA25 in the siRNA screen, which led us to consider whether pharmacological inhibition of Mre11 would potentiate PA25 antiviral activity. Since Mre11 is generally regarded as both a sensor and central mediator of DSB repair 
, our data strongly argue that PA25 initiates HPV episome elimination by introducing DSBs within the HPV episome. The altered regulation of numerous other DSB repair genes by PA25 including ATM, CHEK2, RAD1, and XRCC4 is fully consistent with this idea ().
Drugs that act within DDR pathways to sensitize cells to radiation or to chemotherapeutic agents have the potential to enhance and extend cancer therapy by magnifying the toxic effect within the targeted cells 
. These approaches work by preventing repair and allowing the accumulation of extensive DNA damage that proves toxic to the targeted cells. Mirin is an inhibitor of Mre11-associated exonuclease activity that was identified in a forward genetic screen, and subsequently shown to block the ability of MRN to repair DNA via HR 
. We show here that Mirin makes viral episomes more susceptible to PA25 in a manner that is analogous to radiation and chemotherapeutic sensitizers. This observation was made after Mre11 emerged from the siRNA screen as a repressor of PA25 antiviral activity. These results--that PA25-dependent elimination of episomes is repressed by Mre11 and sensitized by Mirin--provides a powerful argument that PA25 acts to damage viral episomes, causing them to be eliminated from cells. It should be noted that PA25 is not toxic to cells at concentrations vastly exceeding those used in this study, and therefore the effect on episomes is a specific DNA damage event that is primarily manifested by viral DNA elimination rather than cell death 
. The lack of apoptosis genes affected by PA25 is consistent with this concept (, ).
The heterotrimeric Rad9-Hus1-Rad1 (9-1-1) complex is also found to play a role in PA25-dependent loss of HPV episomes. The 9-1-1 complex is a processivity clamp related to PCNA that acts as a scaffold to bring DDR and checkpoint effectors to sites of DNA damage 
. The 9-1-1 complex has been found to associate with and stimulate numerous checkpoint and base excision repair enzymes, and therefore may serve to coordinate DNA repair and checkpoint control 
. RAD1 emerged as the gene most altered by PA25 in expression studies (, ). Rad1 and Rad9 were also both identified in the siRNA screen as highly significant, but oppositional, modulators of PA25 activity: Rad1 acts to repress PA25 activity while Rad9 is an enhancer ( and , ). The binding of hairpin polyamides, such as those employed in this study, is known to expand the minor groove and shrink the major groove of DNA. In contrast, association of the 9-1-1 complex with DNA has the opposite effect, resulting in a contraction of the DNA minor groove and an expansion of the major groove 
. For these reasons the 9-1-1 complex is a good candidate sensor and coordinator of PA25 antiviral activity that may serve to recruit an enzymatic mechanism to the HPV episome resulting in its ultimate destruction. At present it is not understood why Rad1 serves as a PA25 repressor while its 9-1-1 partner Rad9 acts as an enhancer. This difference may be attributable to alterations in 9-1-1 stoichiometry following siRNA treatment, or to differences in their reported DNA binding properties 
The process of studying the PA25 mechanism of action required that the effects of 240 DDR siRNAs also be observed on normal HPV stability in the absence of PA25. HPV16 and HPV31 episome levels fluctuated in cells in response to a panel of DDR siRNAs in a similar manner even though the W12E cell line was derived from a cervical biopsy while the cells maintaining HPV31 were generated in a laboratory from foreskin keratinocytes. The four experiments used in these studies included three experiments (days 1−3) utilizing W12E cells and 1 experiment (day 4) using keratinocytes maintaining HPV31 episomes. A previous report indicated that these genotypes use different modes of DNA replication in cells 
. Using hierarchical clustering () we established that the day 4 (HPV31) data falls within the range of similarity established for days 1−3 (HPV16). Using this approach, multiple HR and FA pathway siRNAs were identified as significant modifiers of normal HPV episome levels (). FANCC and FANCF were found to contribute to HPV episomal stability (). Since the FA and HR pathways were previously implicated in HPV persistence and the viral life cycle 
, these correlations serve as further support for the validity of our studies, and further implicate these pathways as central to the survival and persistence of HPV episomes in cells. Likewise siRNAs for TP53 and MTOR significantly affect HPV episome levels--siRNA to TP53 resulted in an increase in episomes while MTOR knockdown decreased episome levels. Both of these proteins are known targets of HPV E6, which is required for episome maintenance in cells 
A number of novel genes are also identified as highly significant in normal HPV episome maintenance by our siRNA study (). Members of excision repair, mismatch repair, and other DDR pathways are highly significant in the siRNA screen and therefore excellent candidate modifiers of episome stability (). Notably, both TDP1 and TDP2, the only known human genes whose activities are required for removing trapped DNA cleavage complexes of topoisomerase I (TDP1) and topoisomerase II (TDP2) 
, were both identified as regulators of HPV episome stability. TDP1 knockdown causes a net gain of episomes while TDP2 knockdown results in a net loss (). These genes were originally identified by their ability to remove topoisomerase cleavage complexes created in the presence of topoisomerase poisons. However, there is a growing appreciation that DNA metabolic events including DNA damage, ssDNA breaks, and misinsertion of ribonucleotides can trap topoisomerase I, while transcription-related events and abasic sites may cause trapping of topoisomerase II 
. We therefore hypothesize that the trapping of both topoisomerase I and II on HPV episomes are events that must be overcome for successful HPV episome maintenance. Together, these observations from the siRNA screen make a significant contribution to an expanding knowledge base that seeks to understand the role of DDR pathways in normal HPV episome maintenance.
It is apparent that HPV episome stability can be greatly diminished under certain conditions. Here we show a role for multiple DDR genes in mediating the loss of HPV DNA in response to PA25. It was previously shown that the ATR/Chk1 pathway does not play a role in PA25-mediated episome loss, yet does have its own role in controlling viral DNA stability 
. However, it should be noted that it remains to be determined in both cases how viral DNA destruction is executed by the cell. We have shown here that the 9-1-1 complex is a candidate sensor that may play a role in calling for HPV episome removal, but the elements that mediate viral DNA destruction remain unknown. Likewise, Mre11 and MRN appear to play a role in stabilizing episomes against PA25-dependant damage, but the mediators of viral DNA destruction downstream of Mre11 remain to be determined. We believe that understanding the genes and processes that mediate HPV DNA instability and destruction has the potential to inform us about such diverse topics as viral persistence, interferon-mediated viral DNA loss, and antiviral therapy. Finally, it is interesting to consider the application of similar strategies to other DNA viruses that maintain their genomes as extrachromosomal plasmids.