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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

DNA replication initiated from the integrated HPV origin generates various low-molecular-weight DNA products.High-molecular-weight (HMW) DNA and low-molecular-weight (LMW) DNA were fractionated and purified by Hirt lysis 24 hrs post-transfection from HeLa cells (A) and from SiHa cells (B) that were both transfected as follows: mock-transfection (lanes 1 and 5); 5 µg of HPV18 E1 expression plasmid (lanes 2 and 6); 2 µg of E2 expression plasmid (lanes 3 and 7); 5 µg of HPV18 E1 and 2 µg of E2 expression plasmids (lanes 4 and 8). A 3 µg portion of HMW DNA and three times the respective amount of LMW DNA were digested with HindIII (A) or Acc65I/BshTI (B) and separated on a one-dimensional gel. The integrated HPV URR-specific signals were detected by Southern blot analysis. (C) Schematic presentation of the migration of dsDNA linear, supercoiled, and open circle molecules on 2D neutral-neutral gels. (D, E) 5 µg of HPV18 E1 and 2 µg of E2 expression plasmids were transfected into HeLa cells and LMW DNA was purified by Hirt lysis 48 hrs post-transfection. Extracted DNA was digested with DpnI and fractionated by the CsCl-ethidium bromide density gradient. The fraction of linear fragments (lin) and open circular molecules (oc) (D) and the fraction of supercoiled circular plasmids (sc) (E) were separated on a 2D gel, transferred to a nylon filter, and probed with the HPV18 genomic fragment (from nt 3917 to1575). Numbers shown on the axes represent the markers of linear (lin) and supercoiled circular (sc) DNA forms. Black arrowhead indicates the shift that was caused by mtDNA. (F) Schematic presentation of HPV16 integration locus within chromosome 13 in SiHa cells, where the cleavage sites of Acc65I, Eco91I, BshTI, and BcuI as well as their distances from HPV origin are presented. (G) Schematic presentation of the migration of replication forks and replication puffs on 2D neutral-neutral gels. (H,I) SiHa cells were co-transfected with 5 µg of HPV18 E1 and 2 µg of E2 expression plasmids. LMW DNA was extracted 24 hrs post-transfection and digested with Acc65I-BshTI (H) and Eco91I-BcuI (I). Respective HPV16 genome fragments were used as probes on 2D Southern blots. (J) SiHa cells were co-transfected with 1 µg of HPV18 E1 and 2 µg of E2 expression plasmids. Extracted LMW DNA was digested with Eco91I-BcuI and analyzed by 2D Southern blots.
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ppat-1000397-g001: DNA replication initiated from the integrated HPV origin generates various low-molecular-weight DNA products.High-molecular-weight (HMW) DNA and low-molecular-weight (LMW) DNA were fractionated and purified by Hirt lysis 24 hrs post-transfection from HeLa cells (A) and from SiHa cells (B) that were both transfected as follows: mock-transfection (lanes 1 and 5); 5 µg of HPV18 E1 expression plasmid (lanes 2 and 6); 2 µg of E2 expression plasmid (lanes 3 and 7); 5 µg of HPV18 E1 and 2 µg of E2 expression plasmids (lanes 4 and 8). A 3 µg portion of HMW DNA and three times the respective amount of LMW DNA were digested with HindIII (A) or Acc65I/BshTI (B) and separated on a one-dimensional gel. The integrated HPV URR-specific signals were detected by Southern blot analysis. (C) Schematic presentation of the migration of dsDNA linear, supercoiled, and open circle molecules on 2D neutral-neutral gels. (D, E) 5 µg of HPV18 E1 and 2 µg of E2 expression plasmids were transfected into HeLa cells and LMW DNA was purified by Hirt lysis 48 hrs post-transfection. Extracted DNA was digested with DpnI and fractionated by the CsCl-ethidium bromide density gradient. The fraction of linear fragments (lin) and open circular molecules (oc) (D) and the fraction of supercoiled circular plasmids (sc) (E) were separated on a 2D gel, transferred to a nylon filter, and probed with the HPV18 genomic fragment (from nt 3917 to1575). Numbers shown on the axes represent the markers of linear (lin) and supercoiled circular (sc) DNA forms. Black arrowhead indicates the shift that was caused by mtDNA. (F) Schematic presentation of HPV16 integration locus within chromosome 13 in SiHa cells, where the cleavage sites of Acc65I, Eco91I, BshTI, and BcuI as well as their distances from HPV origin are presented. (G) Schematic presentation of the migration of replication forks and replication puffs on 2D neutral-neutral gels. (H,I) SiHa cells were co-transfected with 5 µg of HPV18 E1 and 2 µg of E2 expression plasmids. LMW DNA was extracted 24 hrs post-transfection and digested with Acc65I-BshTI (H) and Eco91I-BcuI (I). Respective HPV16 genome fragments were used as probes on 2D Southern blots. (J) SiHa cells were co-transfected with 1 µg of HPV18 E1 and 2 µg of E2 expression plasmids. Extracted LMW DNA was digested with Eco91I-BcuI and analyzed by 2D Southern blots.

Mentions: We have previously demonstrated that HPV replication proteins E1 and E2, which are expressed from the heterologous expression vectors, can induce over-amplification of the integrated HPV origin, which leads to the chromosomal instability of the HPV positive cancer cells [30]. In the initial studies, we used regular one-dimensional agarose gels for the separation and detection of the integrated HPV replication products, followed by hybridization of the Southern blots with sequence-specific probes. In the current study, we opted to use two-dimensional agarose gels to further characterize the different molecular species generated at the integrated HPV locus as a result of the replication or the action of the cellular repair-recombination machinery. First, we enriched the samples for the replication intermediates of the integrated HPV by using the Hirt extraction method [38]. A considerable part of the replicated HPV DNA appears in the low molecular weight (LMW) fraction of the Hirt extracts (Figure 1A and 1B, lanes 8), while there is essentially no signal for unreplicated HPV (Figure 1A and 1B, lanes 5–7). In this experiment, HeLa cells (1A) and SiHa cells (1B) were transfected either with carrier DNA (lanes 1 and 5), HPV18 E1 expression vector alone (lanes 2 and 6), HPV18 E2 expression vector alone (lanes 3 and 7), or with HPV18 E1 and E2 expression vectors together (lanes 4 and 8). Low molecular weight (LMW) and high molecular weight (HMW) DNA fractions were separated on one-dimensional agarose gel and analyzed by Southern blot with HPV18 (1A) or HPV16 (1B) URR-specific probes. Quantification showed that over 50% of the replication signal of integrated HPV (Figure 1A and 1B, compare lanes 4 and 8) can be found in the Hirt LMW extract compared to 5% of the unreplicated DNA of the integrated virus (Figure 1A and 1B, compare lanes 1–3 with 5–7).


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)

DNA replication initiated from the integrated HPV origin generates various low-molecular-weight DNA products.High-molecular-weight (HMW) DNA and low-molecular-weight (LMW) DNA were fractionated and purified by Hirt lysis 24 hrs post-transfection from HeLa cells (A) and from SiHa cells (B) that were both transfected as follows: mock-transfection (lanes 1 and 5); 5 µg of HPV18 E1 expression plasmid (lanes 2 and 6); 2 µg of E2 expression plasmid (lanes 3 and 7); 5 µg of HPV18 E1 and 2 µg of E2 expression plasmids (lanes 4 and 8). A 3 µg portion of HMW DNA and three times the respective amount of LMW DNA were digested with HindIII (A) or Acc65I/BshTI (B) and separated on a one-dimensional gel. The integrated HPV URR-specific signals were detected by Southern blot analysis. (C) Schematic presentation of the migration of dsDNA linear, supercoiled, and open circle molecules on 2D neutral-neutral gels. (D, E) 5 µg of HPV18 E1 and 2 µg of E2 expression plasmids were transfected into HeLa cells and LMW DNA was purified by Hirt lysis 48 hrs post-transfection. Extracted DNA was digested with DpnI and fractionated by the CsCl-ethidium bromide density gradient. The fraction of linear fragments (lin) and open circular molecules (oc) (D) and the fraction of supercoiled circular plasmids (sc) (E) were separated on a 2D gel, transferred to a nylon filter, and probed with the HPV18 genomic fragment (from nt 3917 to1575). Numbers shown on the axes represent the markers of linear (lin) and supercoiled circular (sc) DNA forms. Black arrowhead indicates the shift that was caused by mtDNA. (F) Schematic presentation of HPV16 integration locus within chromosome 13 in SiHa cells, where the cleavage sites of Acc65I, Eco91I, BshTI, and BcuI as well as their distances from HPV origin are presented. (G) Schematic presentation of the migration of replication forks and replication puffs on 2D neutral-neutral gels. (H,I) SiHa cells were co-transfected with 5 µg of HPV18 E1 and 2 µg of E2 expression plasmids. LMW DNA was extracted 24 hrs post-transfection and digested with Acc65I-BshTI (H) and Eco91I-BcuI (I). Respective HPV16 genome fragments were used as probes on 2D Southern blots. (J) SiHa cells were co-transfected with 1 µg of HPV18 E1 and 2 µg of E2 expression plasmids. Extracted LMW DNA was digested with Eco91I-BcuI and analyzed by 2D Southern blots.
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Related In: Results  -  Collection

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

ppat-1000397-g001: DNA replication initiated from the integrated HPV origin generates various low-molecular-weight DNA products.High-molecular-weight (HMW) DNA and low-molecular-weight (LMW) DNA were fractionated and purified by Hirt lysis 24 hrs post-transfection from HeLa cells (A) and from SiHa cells (B) that were both transfected as follows: mock-transfection (lanes 1 and 5); 5 µg of HPV18 E1 expression plasmid (lanes 2 and 6); 2 µg of E2 expression plasmid (lanes 3 and 7); 5 µg of HPV18 E1 and 2 µg of E2 expression plasmids (lanes 4 and 8). A 3 µg portion of HMW DNA and three times the respective amount of LMW DNA were digested with HindIII (A) or Acc65I/BshTI (B) and separated on a one-dimensional gel. The integrated HPV URR-specific signals were detected by Southern blot analysis. (C) Schematic presentation of the migration of dsDNA linear, supercoiled, and open circle molecules on 2D neutral-neutral gels. (D, E) 5 µg of HPV18 E1 and 2 µg of E2 expression plasmids were transfected into HeLa cells and LMW DNA was purified by Hirt lysis 48 hrs post-transfection. Extracted DNA was digested with DpnI and fractionated by the CsCl-ethidium bromide density gradient. The fraction of linear fragments (lin) and open circular molecules (oc) (D) and the fraction of supercoiled circular plasmids (sc) (E) were separated on a 2D gel, transferred to a nylon filter, and probed with the HPV18 genomic fragment (from nt 3917 to1575). Numbers shown on the axes represent the markers of linear (lin) and supercoiled circular (sc) DNA forms. Black arrowhead indicates the shift that was caused by mtDNA. (F) Schematic presentation of HPV16 integration locus within chromosome 13 in SiHa cells, where the cleavage sites of Acc65I, Eco91I, BshTI, and BcuI as well as their distances from HPV origin are presented. (G) Schematic presentation of the migration of replication forks and replication puffs on 2D neutral-neutral gels. (H,I) SiHa cells were co-transfected with 5 µg of HPV18 E1 and 2 µg of E2 expression plasmids. LMW DNA was extracted 24 hrs post-transfection and digested with Acc65I-BshTI (H) and Eco91I-BcuI (I). Respective HPV16 genome fragments were used as probes on 2D Southern blots. (J) SiHa cells were co-transfected with 1 µg of HPV18 E1 and 2 µg of E2 expression plasmids. Extracted LMW DNA was digested with Eco91I-BcuI and analyzed by 2D Southern blots.
Mentions: We have previously demonstrated that HPV replication proteins E1 and E2, which are expressed from the heterologous expression vectors, can induce over-amplification of the integrated HPV origin, which leads to the chromosomal instability of the HPV positive cancer cells [30]. In the initial studies, we used regular one-dimensional agarose gels for the separation and detection of the integrated HPV replication products, followed by hybridization of the Southern blots with sequence-specific probes. In the current study, we opted to use two-dimensional agarose gels to further characterize the different molecular species generated at the integrated HPV locus as a result of the replication or the action of the cellular repair-recombination machinery. First, we enriched the samples for the replication intermediates of the integrated HPV by using the Hirt extraction method [38]. A considerable part of the replicated HPV DNA appears in the low molecular weight (LMW) fraction of the Hirt extracts (Figure 1A and 1B, lanes 8), while there is essentially no signal for unreplicated HPV (Figure 1A and 1B, lanes 5–7). In this experiment, HeLa cells (1A) and SiHa cells (1B) were transfected either with carrier DNA (lanes 1 and 5), HPV18 E1 expression vector alone (lanes 2 and 6), HPV18 E2 expression vector alone (lanes 3 and 7), or with HPV18 E1 and E2 expression vectors together (lanes 4 and 8). Low molecular weight (LMW) and high molecular weight (HMW) DNA fractions were separated on one-dimensional agarose gel and analyzed by Southern blot with HPV18 (1A) or HPV16 (1B) URR-specific probes. Quantification showed that over 50% of the replication signal of integrated HPV (Figure 1A and 1B, compare lanes 4 and 8) can be found in the Hirt LMW extract compared to 5% of the unreplicated DNA of the integrated virus (Figure 1A and 1B, compare lanes 1–3 with 5–7).

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