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Integration of the human papillomavirus (HPV) genome into host cell chromosomes has been observed in a majority of HPV-positive cervical cancers and a subset of oral HPV-associated cancers. HPV integration also occurs in long-term cell culture. Screening for HPV integration can be labor intensive and yield results that are difficult to interpret. Here we describe an assay based on exonuclease V (ExoV/RecBCD) and quantitative polymerase chain reaction (qPCR) to determine if samples from cell lines and tissues contain episomal or integrated HPV. This assay can be applied to screen other small DNA viruses with episomal/linear genome configurations in their viral lifecycle and has the potential to be used in clinical settings to define viral genomic conformations associated with disease. © 2020 Wiley Periodicals LLC. Basic Protocol: Exonuclease V genomic DNA digestion and qPCR for detection of HPV16 genome configuration in cells Support Protocol: Exonuclease V analysis of HPV16 genome configuration in tissues Alternate Protocol: Determining HPV integration type or integrity of HPV episome.

BACKGROUND Cellular mitochondrial DNA (mtDNA) is organized as circular, covalently closed and double-stranded DNA. Studies have demonstrated the presence of short mtDNA fragments in plasma. It is not known whether circular mtDNA might concurrently exist with linear mtDNA in plasma. METHODS We elucidated the topology of plasma mtDNA using restriction enzyme BfaI cleavage signatures on mtDNA fragment ends to differentiate linear and circular mtDNA. mtDNA fragments with both ends carrying BfaI cleavage signatures were defined as circular-derived mtDNA, whereas those with no cleavage signature or with 1 cleavage signature were defined as linear-derived mtDNA. An independent assay using exonuclease V to remove linear DNA followed by restriction enzyme MspI digestion was used for confirming the conclusions based on BfaI cleavage analysis. We analyzed the presence of BfaI cleavage signatures on plasma DNA ends in nonhematopoietically and hematopoietically derived DNA molecules by sequencing plasma DNA of patients with liver transplantation and bone marrow transplantation. RESULTS Both linear and circular mtDNA coexisted in plasma. In patients with liver transplantation, donor-derived (i.e., liver) mtDNA molecules were mainly linear (median fraction, 91%; range, 75%–97%), whereas recipient-derived (i.e., hematopoietic) mtDNA molecules were mainly circular (median fraction, 88%; range, 77%–93%). The proportion of linear mtDNA was well correlated with liver DNA contribution in the plasma DNA pool (r = 0.83; P value = 0.0008). Consistent data were obtained from a bone marrow transplantation recipient in whom the donor-derived (i.e., hematopoietic) mtDNA molecules were predominantly circular. CONCLUSIONS Linear and circular mtDNA molecules coexist in plasma and may have different tissue origins.

Preparation of an in vitro coupled transcription-translation system from E. coli strains lacking exonuclease V has greatly improved the system for use with added linear DNA fragments. In fact, in extracts of these mutants linear fragments are stable for several hours. However the cell extracts show a high level of endogenous background. To avoid this complication extracts were prepared at 30 degrees C from a mutant carrying a temperature-sensitive exonuclease V. Polypeptides coded by a specific DNA region, e.g., delineated by restriction endonuclease sites, can now be easily identified.

The ATP-dependent exonuclease V from Micrococcus lysodekticus shows a Michaelian relation between steady-state velocity and the concentration of T7 DNA substrate. The Km, (expressed as a mass concentration) does not change when the T7 DNA is broken into smaller fragments by a restriction enzyme. This is interpreted to mean that the predominant process by which the exonuclease-V–DNA complex breaks down is digestion of the entire DNA molecule rather than physical dissociation, in accord with the already known processive nature of degradation by this enzyme. The way that the V and Km, towards DNA vary with ATP and ADP concentration suggests that enzyme-DNA complex is predominantly formed by reaction of DNA with an enzyme-ATP complex rather than with bare enzyme.

There appear to be three pathways of recombination active or potentially active in strains of Escherichia coli (Clark, 1971, 1973). The operation of two of these three pathways has so far been associated with nuclease activities. The RecBC pathway makes use of exonuclease V (ExoV), the product of the recB and recC genes (see e.g. Goldmark and Linn, 1972). The RecE pathway does not use ExoV; it proceeds in cells that contain exonuclease VIII (ExoVIII). The RecF pathway also does not use ExoV. The RecF pathway appears to be inhibited by exonuclease I (Exol) in that it operates at high efficiency only when Exol is mutationally inactivated by sbcB mutations. Here we wish to consider the role that ExoVIII may play in the RecE pathway, particularly in comparison with the roles that can be postulated for ExoV in the RecBC pathway and for the exonuclease of lambda (Exoλ) in the Red recombination system of lambda.