Beta human papillomavirus 8 E6 allows colocalization of non-homologous end joining and homologous recombination repair factors

Abstract

Beta human papillomavirus (β-HPV) are hypothesized to make DNA damage more mutagenic and potentially more carcinogenic. Double strand breaks (DSBs) are the most deleterious DNA lesion. They are typically repaired by homologous recombination (HR) or non-homologous end joining (NHEJ). HR occurs after DNA replication while NHEJ can occur at any point in the cell cycle. HR and NHEJ are not thought to occur in the same cell at the same time. HR is restricted to cells in phases of the cell cycle where homologous templates are available, while NHEJ occurs primarily during G1. β-HPV type 8 protein E6 (8E6) attenuates both repair pathways. We use a series of immunofluorescence microscopy and flow cytometry experiments to better define the impact of this attenuation. We found that 8E6 causes colocalization of HR factors (RPA70 and RAD51) with an NHEJ factor (activated DNA-PKcs or pDNA-PKcs) at persistent DSBs. 8E6 also causes RAD51 foci to form during G1. The initiation of NHEJ and HR at the same lesion could lead to antagonistic DNA end processing. Further, HR cannot be readily completed in an error-free manner during G1. Both aberrant repair events would cause deletions. To determine if these mutations were occurring, we used next generation sequencing of the 200kb surrounding a CAS9-induced DSB. 8E6 caused a 21-fold increase in deletions. Chemical and genetic inhibition of p300 as well as an 8E6 mutant that is incapable of destabilizing p300 demonstrates that 8E6 is acting via p300 destabilization. More specific chemical inhibitors of DNA repair provided mechanistic insight by mimicking 8E6-induced dysregulation of DNA repair in a virus-free system. Specifically, inhibition of NHEJ causes RAD51 foci to form in G1 and colocalization of RAD51 with pDNA-PKcs.

Beta HPV Deregulates Double-Strand Break Repair

“…Instead, they delay the resolution of RAD51 foci, suggesting that the repair complexes that form may be non-functional [ 84 ]. Supporting this idea, some of the RAD51 foci that form in cells expressing HPV8 E6 occur in G1 phase, when they are unlikely to be efficiently resolved due to the lack of a homologous template [ 86 ]. HPV38 E6 that weakly binds to p300 did not significantly decrease BRCA1 or BRCA2 expression.…”

“…To address this knowledge gap, a recent effort tracked the persistent HR and NHEJ foci found in earlier work [ 84 , 99 ]. This work found that HPV8 E6 caused HR factors (RPA70 and RAD51) to be recruited to sites of stalled NHEJ repair [ 86 ]. NHEJ and HR are intrinsically incompatible.…”

“…This is expected to lead to increased mutations, especially deletions. Next-generation sequencing targeted at 200 kb surrounding a CAS9-induced DSB supports this idea, by showing a 20-fold increase in deletions in cells with HPV8 E6 compared to vector control cells [ 86 ]. Our unpublished data show that HPV8 E6 increases the use of a backup DSB repair pathway, known as alternative end-joining (Alt-EJ).…”

“…Radiation mimic reagent zeocin was used to induce DSBs to study how HPV8 E6 deregulates NHEJ [ 99 ]. sgRNA/CAS9 was used to induce DSB at a single genomic locus in cells expressing HPV8 E6 [ 86 , 114 ]. Their ability to cause DSBs in different manners can be used to examine alterations in DSB repair caused by beta HPVs.…”

“…Thus, this approach has the most untapped potential of the techniques discussed in this review. One potential use would be to expand on our previous observation that HPV8 E6 promotes colocalization of NHEJ and HR factors [ 86 ]. Live-cell imaging would a definitive determination of how these abnormal complexes form and whether they are capable of resolving a DSB or marking cells for the death that is associated with an unrepaired DSB [ 133 , 134 ].…”