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DNA moves sequentially towards the nuclear matrix during DNA replication in vivo.

Rivera-Mulia JC, Hernández-Muñoz R, Martínez F, Aranda-Anzaldo A - BMC Cell Biol. (2011)

Bottom Line: We have previously determined in quiescent rat hepatocytes that a 162 kbp genomic region containing members of the albumin gene family is organized into five structural DNA loops.Looped DNA moves in a sequential fashion, as if reeled in, towards the NM during DNA replication in vivo thus supporting the notion that the DNA template is pulled progressively towards the replication factories on the NM so as to be replicated.These results provide further evidence that the structural DNA loops correspond to the actual replicons in vivo.

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Affiliation: Laboratorio de Biología Molecular, Facultad de Medicina, Universidad Autónoma del Estado de México, Apartado Postal 428, CP 50000 Toluca, Edo Méx, México.

ABSTRACT

Background: In the interphase nucleus of metazoan cells DNA is organized in supercoiled loops anchored to a nuclear matrix (NM). There is varied evidence indicating that DNA replication occurs in replication factories organized upon the NM and that DNA loops may correspond to the actual replicons in vivo. In normal rat liver the hepatocytes are arrested in G0 but they synchronously re-enter the cell cycle after partial-hepatectomy leading to liver regeneration in vivo. We have previously determined in quiescent rat hepatocytes that a 162 kbp genomic region containing members of the albumin gene family is organized into five structural DNA loops.

Results: In the present work we tracked down the movement relative to the NM of DNA sequences located at different points within such five structural DNA loops during the S phase and after the return to cellular quiescence during liver regeneration. Our results indicate that looped DNA moves sequentially towards the NM during replication and then returns to its original position in newly quiescent cells, once the liver regeneration has been achieved.

Conclusions: Looped DNA moves in a sequential fashion, as if reeled in, towards the NM during DNA replication in vivo thus supporting the notion that the DNA template is pulled progressively towards the replication factories on the NM so as to be replicated. These results provide further evidence that the structural DNA loops correspond to the actual replicons in vivo.

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Positional mapping relative to the NM using a higher DNA template concentration. Positional mapping relative to the NM of specific target sequences along the 162 kbp rat albumin gene-family genomic region by PCR using a higher DNA template concentration. Nucleoids from G0 rat hepatocytes were treated with DNase I (0.5 U/ml) for different times (Figure 2A). The residual NM-bound DNA in the partially digested samples was directly used as template for PCR amplification of the chosen target sequences (a - o). For these experiments the nuclear matrix-bound DNA template was increased six-fold (from 10 to 60 ng) and the DNA polymerase concentration was doubled (from 0.7 to 1.25 U) in order to facilitate the amplification of target sequences embedded within the NM. The specific amplicons were resolved in 2% agarose gels and stained with ethidium bromide (0.5 μg/ml). C, 0' digestion-time control. The amplicons were scored either as positive or negative as a function of endonuclease digestion time and for each topological zone relative to the NM, depending on whether or not they were detected by a digital image-analysis system (Kodak 1D Image Analysis Software 3.5) using the default settings. Topological zones relative to the NM: D, distal; P, proximal; VC, very close; E, embedded within the NM. (-) Negative control (no template); (+) positive control (pure genomic DNA as template). For detailed analysis of results see Table 4.
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Figure 6: Positional mapping relative to the NM using a higher DNA template concentration. Positional mapping relative to the NM of specific target sequences along the 162 kbp rat albumin gene-family genomic region by PCR using a higher DNA template concentration. Nucleoids from G0 rat hepatocytes were treated with DNase I (0.5 U/ml) for different times (Figure 2A). The residual NM-bound DNA in the partially digested samples was directly used as template for PCR amplification of the chosen target sequences (a - o). For these experiments the nuclear matrix-bound DNA template was increased six-fold (from 10 to 60 ng) and the DNA polymerase concentration was doubled (from 0.7 to 1.25 U) in order to facilitate the amplification of target sequences embedded within the NM. The specific amplicons were resolved in 2% agarose gels and stained with ethidium bromide (0.5 μg/ml). C, 0' digestion-time control. The amplicons were scored either as positive or negative as a function of endonuclease digestion time and for each topological zone relative to the NM, depending on whether or not they were detected by a digital image-analysis system (Kodak 1D Image Analysis Software 3.5) using the default settings. Topological zones relative to the NM: D, distal; P, proximal; VC, very close; E, embedded within the NM. (-) Negative control (no template); (+) positive control (pure genomic DNA as template). For detailed analysis of results see Table 4.

Mentions: Considering that the topology-dependent resistance to DNase I of target sequences is relative but not absolute, the extinction of a target amplicon at a given DNA-digestion time corresponds to the actual reduction of the target template to a non-amplifiable concentration within the nucleoid population analyzed. Moreover, DNA embedded within the NM is poorly accessible to externally added polymerases and so it is non- amplifiable under our standard experimental conditions. Nevertheless, a six-fold rise in the concentration of NM-bound DNA template (from 10 to 60 ng) allows detection of some target sequences in the NM-embedded fraction even in control G0 samples (Figure 6) but this situation leads to a general shift of all target sequences towards closer positions relative to the NM. Moreover, the location of some target sequences drifts between adjacent topological zones from one experiment to another due to the artifactual rise in the relative abundance of the target-sequence templates (Table 4). Since our results correspond to an average snapshot of the S phase in vivo the loop DNA configuration depicted (Figure 5B) is very likely the sum of different hepatocyte subpopulations at different stages within the S phase. Therefore, under our standard experimental conditions only those nucleoids subpopulations in which the target sequence is not actually embedded within the NM may contain the corresponding amplifiable template. Moreover, LARs themselves cannot be reliable targets for PCR amplification on NM- bound templates since they usually contain repetitive sequences that preclude the design of highly-specific and efficient primers [23] but they are also poorly accessible for amplification by being embedded within the NM.


DNA moves sequentially towards the nuclear matrix during DNA replication in vivo.

Rivera-Mulia JC, Hernández-Muñoz R, Martínez F, Aranda-Anzaldo A - BMC Cell Biol. (2011)

Positional mapping relative to the NM using a higher DNA template concentration. Positional mapping relative to the NM of specific target sequences along the 162 kbp rat albumin gene-family genomic region by PCR using a higher DNA template concentration. Nucleoids from G0 rat hepatocytes were treated with DNase I (0.5 U/ml) for different times (Figure 2A). The residual NM-bound DNA in the partially digested samples was directly used as template for PCR amplification of the chosen target sequences (a - o). For these experiments the nuclear matrix-bound DNA template was increased six-fold (from 10 to 60 ng) and the DNA polymerase concentration was doubled (from 0.7 to 1.25 U) in order to facilitate the amplification of target sequences embedded within the NM. The specific amplicons were resolved in 2% agarose gels and stained with ethidium bromide (0.5 μg/ml). C, 0' digestion-time control. The amplicons were scored either as positive or negative as a function of endonuclease digestion time and for each topological zone relative to the NM, depending on whether or not they were detected by a digital image-analysis system (Kodak 1D Image Analysis Software 3.5) using the default settings. Topological zones relative to the NM: D, distal; P, proximal; VC, very close; E, embedded within the NM. (-) Negative control (no template); (+) positive control (pure genomic DNA as template). For detailed analysis of results see Table 4.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3037911&req=5

Figure 6: Positional mapping relative to the NM using a higher DNA template concentration. Positional mapping relative to the NM of specific target sequences along the 162 kbp rat albumin gene-family genomic region by PCR using a higher DNA template concentration. Nucleoids from G0 rat hepatocytes were treated with DNase I (0.5 U/ml) for different times (Figure 2A). The residual NM-bound DNA in the partially digested samples was directly used as template for PCR amplification of the chosen target sequences (a - o). For these experiments the nuclear matrix-bound DNA template was increased six-fold (from 10 to 60 ng) and the DNA polymerase concentration was doubled (from 0.7 to 1.25 U) in order to facilitate the amplification of target sequences embedded within the NM. The specific amplicons were resolved in 2% agarose gels and stained with ethidium bromide (0.5 μg/ml). C, 0' digestion-time control. The amplicons were scored either as positive or negative as a function of endonuclease digestion time and for each topological zone relative to the NM, depending on whether or not they were detected by a digital image-analysis system (Kodak 1D Image Analysis Software 3.5) using the default settings. Topological zones relative to the NM: D, distal; P, proximal; VC, very close; E, embedded within the NM. (-) Negative control (no template); (+) positive control (pure genomic DNA as template). For detailed analysis of results see Table 4.
Mentions: Considering that the topology-dependent resistance to DNase I of target sequences is relative but not absolute, the extinction of a target amplicon at a given DNA-digestion time corresponds to the actual reduction of the target template to a non-amplifiable concentration within the nucleoid population analyzed. Moreover, DNA embedded within the NM is poorly accessible to externally added polymerases and so it is non- amplifiable under our standard experimental conditions. Nevertheless, a six-fold rise in the concentration of NM-bound DNA template (from 10 to 60 ng) allows detection of some target sequences in the NM-embedded fraction even in control G0 samples (Figure 6) but this situation leads to a general shift of all target sequences towards closer positions relative to the NM. Moreover, the location of some target sequences drifts between adjacent topological zones from one experiment to another due to the artifactual rise in the relative abundance of the target-sequence templates (Table 4). Since our results correspond to an average snapshot of the S phase in vivo the loop DNA configuration depicted (Figure 5B) is very likely the sum of different hepatocyte subpopulations at different stages within the S phase. Therefore, under our standard experimental conditions only those nucleoids subpopulations in which the target sequence is not actually embedded within the NM may contain the corresponding amplifiable template. Moreover, LARs themselves cannot be reliable targets for PCR amplification on NM- bound templates since they usually contain repetitive sequences that preclude the design of highly-specific and efficient primers [23] but they are also poorly accessible for amplification by being embedded within the NM.

Bottom Line: We have previously determined in quiescent rat hepatocytes that a 162 kbp genomic region containing members of the albumin gene family is organized into five structural DNA loops.Looped DNA moves in a sequential fashion, as if reeled in, towards the NM during DNA replication in vivo thus supporting the notion that the DNA template is pulled progressively towards the replication factories on the NM so as to be replicated.These results provide further evidence that the structural DNA loops correspond to the actual replicons in vivo.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratorio de Biología Molecular, Facultad de Medicina, Universidad Autónoma del Estado de México, Apartado Postal 428, CP 50000 Toluca, Edo Méx, México.

ABSTRACT

Background: In the interphase nucleus of metazoan cells DNA is organized in supercoiled loops anchored to a nuclear matrix (NM). There is varied evidence indicating that DNA replication occurs in replication factories organized upon the NM and that DNA loops may correspond to the actual replicons in vivo. In normal rat liver the hepatocytes are arrested in G0 but they synchronously re-enter the cell cycle after partial-hepatectomy leading to liver regeneration in vivo. We have previously determined in quiescent rat hepatocytes that a 162 kbp genomic region containing members of the albumin gene family is organized into five structural DNA loops.

Results: In the present work we tracked down the movement relative to the NM of DNA sequences located at different points within such five structural DNA loops during the S phase and after the return to cellular quiescence during liver regeneration. Our results indicate that looped DNA moves sequentially towards the NM during replication and then returns to its original position in newly quiescent cells, once the liver regeneration has been achieved.

Conclusions: Looped DNA moves in a sequential fashion, as if reeled in, towards the NM during DNA replication in vivo thus supporting the notion that the DNA template is pulled progressively towards the replication factories on the NM so as to be replicated. These results provide further evidence that the structural DNA loops correspond to the actual replicons in vivo.

Show MeSH
Related in: MedlinePlus