Skip to main content
Article thumbnail
Location of Repository

Mechanism of Genomic Instability in Cells Infected with the High-Risk Human Papillomaviruses

By Meelis Kadaja, Helen Isok-Paas, Triin Laos, Ene Ustav and Mart Ustav


In HPV–related cancers, the “high-risk” human papillomaviruses (HPVs) are frequently found integrated into the cellular genome. The integrated subgenomic HPV fragments express viral oncoproteins and carry an origin of DNA replication that is capable of initiating bidirectional DNA re-replication in the presence of HPV replication proteins E1 and E2, which ultimately leads to rearrangements within the locus of the integrated viral DNA. The current study indicates that the E1- and E2-dependent DNA replication from the integrated HPV origin follows the “onion skin”–type replication mode and generates a heterogeneous population of replication intermediates. These include linear, branched, open circular, and supercoiled plasmids, as identified by two-dimensional neutral-neutral gel-electrophoresis. We used immunofluorescence analysis to show that the DNA repair/recombination centers are assembled at the sites of the integrated HPV replication. These centers recruit viral and cellular replication proteins, the MRE complex, Ku70/80, ATM, Chk2, and, to some extent, ATRIP and Chk1 (S317). In addition, the synthesis of histone γH2AX, which is a hallmark of DNA double strand breaks, is induced, and Chk2 is activated by phosphorylation in the HPV–replicating cells. These changes suggest that the integrated HPV replication intermediates are processed by the activated cellular DNA repair/recombination machinery, which results in cross-chromosomal translocations as detected by metaphase FISH. We also confirmed that the replicating HPV episomes that expressed the physiological levels of viral replication proteins could induce genomic instability in the cells with integrated HPV. We conclude that the HPV replication origin within the host chromosome is one of the key factors that triggers the development of HPV–associated cancers. It could be used as a starting point for the “onion skin”–type of DNA replication whenever the HPV plasmid exists in the same cell, which endangers the host genomic integrity during the initial integration and after the de novo infection

Topics: Research Article
Publisher: Public Library of Science
OAI identifier:
Provided by: PubMed Central
Download PDF:
Sorry, we are unable to provide the full text but you may find it at the following location(s):
  • http://www.pubmedcentral.nih.g... (external link)
  • Suggested articles


    1. (2003). A p53-dependent checkpoint pathway prevents rereplication.
    2. (2006). A phosphorylation map of the bovine papillomavirus E1 helicase.
    3. (2004). Acquisition of high-level chromosomal instability is associated with integration of human papillomavirus type 16 in cervical keratinocytes.
    4. (2000). Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro.
    5. (2002). Changes in cervical keratinocyte gene expression associated with integration of human papillomavirus 16.
    6. (1980). Characteristics of an SV40-plasmid recombinant and its movement into and out of the genome of a murine cell.
    7. (2008). Characterization of naturally occurring HPV16 integration sites isolated from cervical keratinocytes under noncompetitive conditions.
    8. (2002). Chk2 activation and phosphorylationdependent oligomerization.
    9. (2006). Chromatin structural elements and chromosomal translocations in leukemia.
    10. (1996). Cis and trans requirements for stable episomal maintenance of the BPV-1 replicator.
    11. (1995). cis-Acting components of human papillomavirus (HPV) DNA replication: linker substitution analysis of the HPV type 11 origin.
    12. (1994). Coexistence of episomal and integrated HPV16 DNA in squamous cell carcinoma of the cervix.
    13. (1999). Comprehensive and definitive molecular cytogenetic characterization of HeLa cells by spectral karyotyping.
    14. (2002). Control of DNA replication and chromosome ploidy by geminin and cyclin A.
    15. (1998). Current Protocols in Molecular Biology.
    16. (2007). Daxx mediates SUMOdependent transcriptional control and subnuclear compartmentalization.
    17. (2006). Deregulation of Cdt1 induces chromosomal damage without rereplication and leads to chromosomal instability.
    18. (2006). Differential usage of non-homologous end-joining and homologous recombination in double strand break repair.
    19. (1992). Disruption of either the E1 or the E2 regulatory gene of human papillomavirus type 16 increases viral immortalization capacity.
    20. (2006). DNA damage-induced cell cycle regulation and function of novel Chk2 phosphoresidues.
    21. (1984). DNA sequence studies of simian virus 40 chromosomal excision and integration in rat cells.
    22. (2005). Dutta A
    23. Dutta A (2004) Rereplication by depletion of geminin is seen regardless of p53 status and activates a G2/M checkpoint.
    24. (2000). Dynamics of DNA replication factories in living cells.
    25. (2006). Early integration of high copy HPV16 detectable in women with normal and low grade cervical cytology and histology.
    26. (1991). Episomal and integrated human papillomavirus in cervical neoplasia shown by nonisotopic in situ hybridisation.
    27. (2003). Extrachromosomal circular DNA of tandemly repeated genomic sequence si nD r o s o p h i l a .G e n o m eR e
    28. (2007). Genomic instability of the host cell induced by the human papillomavirus replication machinery.
    29. (1999). Human papillomavirus DNA replication compartments in a transient DNA replication system.
    30. (2003). Human papillomavirus type 16 E2 protein has no effect on transcription from episomal viral DNA.
    31. (2003). Human papillomavirus type 16 E7 oncoprotein can induce abnormal centrosome duplication through a mechanism independent of inactivation of retinoblastoma protein family members.
    32. (2006). Human papillomavirus type 16 integration in cervical carcinoma in situ and in invasive cervical cancer.
    33. (2001). Impaired nonhomologous end-joining provokes soft tissue sarcomas harboring chromosomal translocations, amplifications, and deletions.
    34. (1996). In vitro synthesis of oncogenic human papillomaviruses requires episomal genomes for differentiation-dependent late expression.
    35. (2002). Induction of the bovine papillomavirus origin ‘‘onion skin’’-type DNA replication at high E1 protein concentrations in vivo.
    36. (2003). Initiation of DNA replication: lessons from viral initiator proteins.
    37. (2002). Integrated human papillomavirus type 16 is frequently found in cervical cancer precursors as demonstrated by a novel quantitative real-time PCR technique.
    38. (2007). Integration of high-risk human papillomavirus: a key event in cervical carcinogenesis?
    39. (1995). Integration of human papillomavirus type 16 into the human genome correlates with a selective growth advantage of cells.
    40. (1991). Integration of papillomavirus DNA near myc genes in genital carcinomas and its consequences for proto-oncogene expression.
    41. (2004). Mechanisms of genomic instability in human cancer: insights from studies with human papillomavirus oncoproteins.
    42. (1987). Molecular analysis of integrated human papillomavirus 16 sequences in the cervical cancer cell line SiHa.
    43. (1989). Molecular cloning: a laboratory manual, 2nd ed.
    44. (1990). Mutational analysis of cis elements involved in E2 modulation of human papillomavirus type 16 P97 and type 18 P105 promoters.
    45. (1999). Nucleotide sequences and further characterization of human papillomavirus DNA present in the CaSki, SiHa and HeLa cervical carcinoma cell lines.
    46. (2009). Papillomavirus DNA replication -from initiation to genomic instability.
    47. (2001). Papillomaviruses and Their Replication.
    48. (1999). Physical and genetic mapping of mammalian replication origins.
    49. (2001). Physical state of HPV16 and chromosomal mapping of the integrated form in cervical carcinomas.
    50. (1992). Random-choice replication of extrachromosomal bovine papillomavirus (BPV) molecules in heterogeneous, clonally derived BPV-infected cell lines.
    51. (2008). Regulation of DNA doublestrand break repair pathway choice.
    52. (2007). Replication foci dynamics: replication patterns are modulated by S-phase checkpoint kinases in fission yeast.
    53. (1994). Replication of human papillomavirus (HPV) DNAs supported by the HPV type 18 E1 and E2 proteins.
    54. (2006). Selection of cervical keratinocytes containing integrated HPV16 associates with episome loss and an endogenous antiviral response.
    55. (1967). Selective extraction of polyoma DNA from infected mouse cell cultures.
    56. (2002). Simultaneous mapping of human papillomavirus integration sites and molecular karyotyping in short-term cultures of cervical carcinomas by using 49-color combined binary ratio labeling fluorescence in situ hybridization.
    57. (2000). Simultaneous molecular karyotyping and mapping of viral DNA integration sites by 25-color COBRA-FISH.
    58. (2002). Single copy heterozygote integration of HPV 33 in chromosomal band 5p14 is found in an epithelial cell clone with selective growth advantage.
    59. (1993). Stenlund A
    60. (2007). Structure, dynamics and functions of promyelocytic leukaemia nuclear bodies.
    61. (1979). Studies on simian virus 40 excision from cellular chromosomes.
    62. (2007). The ATR-mediated S phase checkpoint prevents rereplication in mammalian cells when licensing control is disrupted.
    63. (1993). The E1 protein of bovine papilloma virus 1 is an ATP-dependent DNA helicase.
    64. (1992). The E2 binding sites determine the efficiency of replication for the origin of human papillomavirus type 18.
    65. (2008). The endless tale of non-homologous end-joining.
    66. (2002). The human papillomavirus type 16 E6 and E7 oncoproteins independently induce numerical and structural chromosome instability.
    67. (2006). The microarchitecture of DNA replication domains.
    68. (2005). The minor capsid protein L2 contributes to two steps in the human papillomavirus type 31 life cycle.
    69. (1999). The nonhomologous DNA end joining pathway is important for chromosome stability in primary fibroblasts.
    70. (2000). The nonhomologous end-joining pathway of DNA repair is required for genomic stability and the suppression of translocations.
    71. (2003). Two separate replication modes of the bovine papillomavirus BPV1 origin of replication that have different sensitivity to p53.
    72. (2005). Type distribution, viral load and integration status of high-risk human papillomaviruses in pre-stages of cervical cancer (CIN).
    73. (1992). Viral E1 and E2 proteins support replication of homologous and heterologous papillomaviral origins.

    To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.