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Mechanism of genomic instability in cells infected with the high-risk human papillomaviruses.

Kadaja M, Isok-Paas H, Laos T, Ustav E, Ustav M - PLoS Pathog. (2009)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia.

ABSTRACT
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 gammaH2AX, 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.

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Related in: MedlinePlus

Repair of the amplified HPV18 integration locus is coordinated by the ATM and ATR signaling pathways.HeLa cells were transfected and analyzed as described previously. Co-immunostaining of HPV18 E1 (Alexa Fluor 568, second column) and the following proteins are shown at the figure: ATM, Chk2, ATRIP, and Chk1 (S317) (Alexa Fluor 488, first column). Merged images are shown in the third column and DAPI stained nuclei in the fourth column.
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ppat-1000397-g005: Repair of the amplified HPV18 integration locus is coordinated by the ATM and ATR signaling pathways.HeLa cells were transfected and analyzed as described previously. Co-immunostaining of HPV18 E1 (Alexa Fluor 568, second column) and the following proteins are shown at the figure: ATM, Chk2, ATRIP, and Chk1 (S317) (Alexa Fluor 488, first column). Merged images are shown in the third column and DAPI stained nuclei in the fourth column.

Mentions: Eukaryotic cells respond to DNA damage with a rapid activation of signaling cascades that are initiated by the ataxia telangiectasia mutated (ATM) kinase and the ATM and Rad3-related (ATR) kinase. Response to the DSBs, which can be caused by ionizing radiation or radiomimetic drugs, occurs primarily through the ATM-Chk2 signaling pathway. In response to replication fork stalling and other forms of DNA damage that are caused by ultraviolet light, cells activate replication checkpoints, where the central players are the ATR kinase, ATRIP (ATR-interacting protein), and their downstream effector kinase, Chk1. To identify which pathways are activated and recruited to the foci due to the replication of integrated HPV18, we performed co-immunostaining analyses for the HPV18 E1 protein along with ATM, Chk2, ATRIP, or Chk1 proteins in HeLa cells that were transfected with the HPV18 E1 and E2 expression vectors (Figure 5). The results show clear co-localization of ATM and Chk2 at the HPV replication foci, which indicates that a response to DSBs is generated during the HPV replication process. ATRIP also localizes within the HPV replication foci, which demonstrates the presence of RPA-coated ssDNA. In the case of the Chk1 protein, there was only poor co-localization of the Chk1 and E1 proteins when an antibody against phosphorylated Chk1 (S317) was used (Figure 5). There was no co-localization with the E1 protein when the primary antibody against the unphosphorylated form of Chk1 was used (data not shown).


Mechanism of genomic instability in cells infected with the high-risk human papillomaviruses.

Kadaja M, Isok-Paas H, Laos T, Ustav E, Ustav M - PLoS Pathog. (2009)

Repair of the amplified HPV18 integration locus is coordinated by the ATM and ATR signaling pathways.HeLa cells were transfected and analyzed as described previously. Co-immunostaining of HPV18 E1 (Alexa Fluor 568, second column) and the following proteins are shown at the figure: ATM, Chk2, ATRIP, and Chk1 (S317) (Alexa Fluor 488, first column). Merged images are shown in the third column and DAPI stained nuclei in the fourth column.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2666264&req=5

ppat-1000397-g005: Repair of the amplified HPV18 integration locus is coordinated by the ATM and ATR signaling pathways.HeLa cells were transfected and analyzed as described previously. Co-immunostaining of HPV18 E1 (Alexa Fluor 568, second column) and the following proteins are shown at the figure: ATM, Chk2, ATRIP, and Chk1 (S317) (Alexa Fluor 488, first column). Merged images are shown in the third column and DAPI stained nuclei in the fourth column.
Mentions: Eukaryotic cells respond to DNA damage with a rapid activation of signaling cascades that are initiated by the ataxia telangiectasia mutated (ATM) kinase and the ATM and Rad3-related (ATR) kinase. Response to the DSBs, which can be caused by ionizing radiation or radiomimetic drugs, occurs primarily through the ATM-Chk2 signaling pathway. In response to replication fork stalling and other forms of DNA damage that are caused by ultraviolet light, cells activate replication checkpoints, where the central players are the ATR kinase, ATRIP (ATR-interacting protein), and their downstream effector kinase, Chk1. To identify which pathways are activated and recruited to the foci due to the replication of integrated HPV18, we performed co-immunostaining analyses for the HPV18 E1 protein along with ATM, Chk2, ATRIP, or Chk1 proteins in HeLa cells that were transfected with the HPV18 E1 and E2 expression vectors (Figure 5). The results show clear co-localization of ATM and Chk2 at the HPV replication foci, which indicates that a response to DSBs is generated during the HPV replication process. ATRIP also localizes within the HPV replication foci, which demonstrates the presence of RPA-coated ssDNA. In the case of the Chk1 protein, there was only poor co-localization of the Chk1 and E1 proteins when an antibody against phosphorylated Chk1 (S317) was used (Figure 5). There was no co-localization with the E1 protein when the primary antibody against the unphosphorylated form of Chk1 was used (data not shown).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia.

ABSTRACT
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 gammaH2AX, 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.

Show MeSH
Related in: MedlinePlus