Beta human papillomavirus 8 E6 allows colocalization of non-homologous end joining and homologous recombination repair factors
Authors: Hu Changkun , Bugbee Taylor , Palinski Rachel , Wallace Nicholas 
Published: June 2021

AbstractBeta human papillomavirus (β-HPV) are hypothesized to make DNA damage more mutagenic and potentially more carcinogenic. Double strand breaks in DNA (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. They are not thought to occur in the same cell at the same time. By destabilizing p300, β-HPV type 8 protein E6 (β-HPV8 E6) attenuates both repair pathways. However, β-HPV8 E6 delays rather than abrogates DSB repair. Thus, β-HPV8 E6 may cause DSBs to be repaired through a more mutagenic process. To evaluate this, immunofluorescence microscopy was used to detect colocalization, formation, and resolution of DSB repair complexes following damage. Flow cytometry and immunofluorescence microscopy were used to determine the cell cycle distribution of repair complexes. The resulting data show that β-HPV8 E6 causes HR factors (RPA70 and RAD51) to colocalize with a persistent NHEJ factor (pDNA-PKcs). RPA70 complexes gave way to RAD51 complexes as in canonical HR, but RAD51 and pDNA-PKcs colocalization did not resolve within 32 hours of damage. The persistent RAD51 foci occur in G1 phase, consistent with recruitment after NHEJ fails. Chemical inhibition of p300, p300 knockout cells, and an β-HPV8 E6 mutant demonstrate that these phenotypes are the result of β-HPV8 E6-meidated p300 destabilization. Mutations associated with DSB repair were identified using next generation sequencing after a CAS9-induced DSB. β-HPV8 E6 increases the frequency of mutations (>15 fold) and deletions (>20 fold) associated with DSB repair. These data suggest that β-HPV8 E6 causes abnormal DSB repair where both NHEJ and HR occur at the same lesion and that his leads to deletions as the single stranded DNA produced during HR is removed by NHEJ.Author SummaryOur previous work shows that a master transcription regulator, p300, is required for two major DNA double strand break (DSB) repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). By degrading p300, beta genus Human Papillomavirus 8 protein E6 (β-HPV8 E6) hinders DNA-PKcs activity, which is a key factor of NHEJ. β-HPV8 E6 also stalls HR via p300 degradation resulting in the persistence of a core factor, RAD51. NHEJ and HR are known competitive to each other and only one pathway can be initiated to repair a DSB. Particularly, NHEJ tends to be used in G1 phase and HR occurs in S/G2 phase. Here, we show that β-HPV8 E6 allows NHEJ and HR to occur at the same break site. This is expected to be mutagenic because HR generates overhangs while NHEJ removes them. Further, we show that β-HPV8 E6 allows HR to occur in G1, which cannot be finished due to the lack of homologous templates. Finally, our sequencing results show that β-HPV8 E6 significantly increases genomic variations including deletions and insertions following CAS9 induced DSB. This study supports the hypothesis that β-HPV8 infections increases genomic instability.