Once per cell cycle DNA replication in eukaryotic cells is accomplished by temporal separation of the assembly of pre-replication complex (pre-RC) and the actual initiation of DNA synthesis 
. However, the formation of HPV pre-RC that is orchestrated by the viral E1 and E2 proteins can occur simultaneously with the viral DNA synthesis, which allows the HPV origin to be licensed for multiple initiations of DNA replication during a single cell cycle 
. These multiple initiations can effectively complete the DNA replication of the small HPV plasmid at physiological conditions and guarantee its extrachromosomal amplification and maintenance. At the same time, HR-HPV DNA can integrate into the host genome at any time during its episomal maintenance 
, which generates the combination of two HPV origin entities in the same cell – integrated and episomal. Our recent work showed that the integrated HR-HPV origins are effectively mobilized for replication by E1 and E2, which can lead to the generation of irregularity in the genomic DNA 
. Based upon our previous demonstrations, these genomic irregularities are partially resolved in the clonally derived cells, where we found the rearranged tandem repeats of the HPV-host DNA junctions 
. It should be emphasized that similar rearrangements at the loci of integrated HPV are described in W12 cells during the integration of episomal HPV16 
and in SiHa cell lines that are transfected with the HPV16 and HPV18 genomes (). These data indicate that the mobilization of an integrated HPV origin for DNA replication and the subsequent actions of the cellular DNA repair/recombination machinery occur during episomal HPV replication at a physiological level of the replication proteins.
Current analysis of the replication intermediates in SiHa cells show that integrated HPV follows the “onion skin”-type of DNA replication mode. In addition to linear and branched DNA molecules, heterogeneous populations of supercoiled and open circular plasmids were formed. These HPV-origin containing plasmids are most likely the templates for the E1 and E2-driven DNA replication and might be, therefore, one of the mechanisms for gene amplification. Similar chromosomal excision and formation of a heterogeneous pool of circular molecules has also been detected earlier in case of the DNA re-replication of integrated SV40 in the presence of large T antigen 
The structures of DNA breaks that are generated by HPV DNA re-replication should not differ from other types of double strand breaks (DSB), and they should be recognized in eukaryotic cells by either non-homologous end-joining (NHEJ) or homologous recombination (HR). We demonstrated by indirect immunofluorescence that the initiation factors of both NHEJ and HR are localized at the integrated HPV replication centers, which suggests that DSBs are generated by the re-replication of the HPV locus. It is possible that HR, which is the primary DNA repair mechanism in the S-phase, might get saturated by the abundant generation of DSBs during the integrated HPV replication, which would lead to some of the DSBs being repaired by NHEJ. Although the NHEJ machinery plays a significant role in maintaining genome stability and suppressing tumorigenesis 
, it is also responsible for the vast majority of tumorigenic chromosomal translocations. Even the “correct” re-joining of broken ends by NHEJ often results in mutations at the junctions 
. Therefore, NHEJ may primarily contribute to the development of genetic instability that is found in HPV-associated cancers.
Forced assembly of the cellular pre-RC in the S-phase leads to the re-replication of the cellular DNA and the activation of various checkpoint pathways 
. In mammalian cells, the ATR-mediated S-phase checkpoint is immediately activated after accumulation of the RPA-coated ssDNA and before the appearance of DSBs to prevent further DNA re-replication 
. However, our data indicate that ATR is unable to prevent the DNA re-replication from the integrated HPV origin. This allows us to speculate that ATR pathway does not recognize the DNA re-replication that is initiated from the integrated HPV origin, which might be an intrinsic property of the PV replication machinery necessary for the amplification of the viral genome during the initial or late phases of the viral life. The weak localization of ATRIP and Chk1 (S317) to the sites of integrated HPV replication as well as the poor phosphorylation of Chk1 is not sufficient to block the replication. It is possible that the activation of ATR is caused instead by the availability of the RPA-coated ssDNA at the sites of fork collisions and dissociation (). However, if the ATR and ATRIP proteins recognize the sites of integrated HPV replication, the possibilities to inhibit the viral pre-RC might still be limited, since there are only few targets in the viral replication complex that are available for ATR, when compared with the complex initiation mechanisms of the cellular DNA replication. Phosphorylation of the HPV E1 has been extensively studied, but the ability of the ATR to phosphorylate HPV E1 protein has not been demonstrated 
. Additionally, the indirect signaling pathways of ATR through p53 and pRB could be down regulated by the HPV E6 and E7 oncoproteins. It has been also shown that the replication of BPV1 URR reporter plasmid can overcome the inhibitory effect of p53 
. If the prevention of DNA re-replication by ATR fails, the accumulation of DSBs can activate the ATM pathway, as we have concluded from our data. We observed clear localization of ATM and Chk2 to the replication sites of the integrated HPV as well as the clear phosphorylation of Chk2 kinase in the cell population where the replication of the integrated HPV occurred. This indicates that the ATM-Chk2 pathway plays the major role in resolving the DNA damage that is caused by the replication of integrated HPV.
Mechanism of the genomic instability of the cells harboring the replication origin of integrated HPV.
Supported by the observation that Cdt1 is overexpressed in human cancer cells, it has been suggested that DNA re-replication can lead to the chromosomal instability and malignant transformation 
. The current study provides, for the first time, the experimental proof by metaphase FISH that DNA re-replication can indeed lead to chromosomal instability (). Although we used high expression levels of the E1 in this experiment, similar translocations of the co-localized viral-host DNA have been detected in cell lines that were derived from invasive genital carcinomas with native expression levels of the viral proteins 
. In addition, a recent study indicated that local DNA rearrangements occur frequently and shortly after one of the several HPV16 plasmids integrates into W12 cells 
The simultaneous presence of episomal and integrated HPV DNA has been documented in HPV-infected cells, and our data indicate that this dangerous combination can lead to the genomic instability that is driven by the replication of the integrated HPV origin. Such a situation can happen during the primary infection, when one of the hundreds of HPV plasmids accidentally integrates into the host cell genome. Loss of the episomes ultimately generates the cells that carry only the integrated HPV DNA. Such cells can exist in the tissue for a long time and are prone towards the clonal progression to cancer. It is interesting to speculate whether or not these cells can be de novo infected by homologous or heterologous papillomaviruses. Taking into consideration that HPV infections are frequent and wide spread, such a de novo HPV infection could generate a similar situation with the dual status of the HPV genome. Our data allow us to speculate that, in either case, the unscheduled DNA re-replication at the HPV integration locus could be induced, which would provide grounds for the development of genomic instability leading to rearrangements and the formation of the cancer cell. The cellular repair/recombination system is actively involved in this process and is actually the enzymatic machinery that is responsible for introducing the changes into the cellular genome ().