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Histone H1 functions as a stimulatory factor in backup pathways of NHEJ.

Rosidi B, Wang M, Wu W, Sharma A, Wang H, Iliakis G - Nucleic Acids Res. (2008)

Bottom Line: While H1 enhances the DNA-end-joining activities of both DNA Ligase IV and DNA Ligase III, the effect on ligase III is significantly stronger.Histone H1 also enhances the activity of PARP-1.Since histone H1 has been shown to counteract D-NHEJ, these observations and the known functions of the protein identify it as a putative alignment factor operating preferentially within B-NHEJ.

View Article: PubMed Central - PubMed

Affiliation: University of Duisburg-Essen, Medical School, Institute of Medical Radiation Biology, 45122 Essen, Germany.

ABSTRACT
DNA double-strand breaks (DSBs) induced in the genome of higher eukaryotes by ionizing radiation (IR) are predominantly removed by two pathways of non-homologous end-joining (NHEJ) termed D-NHEJ and B-NHEJ. While D-NHEJ depends on the activities of the DNA-dependent protein kinase (DNA-PK) and DNA ligase IV/XRCC4/XLF, B-NHEJ utilizes, at least partly, DNA ligase III/XRCC1 and PARP-1. Using in vitro end-joining assays and protein fractionation protocols similar to those previously applied for the characterization of DNA ligase III as an end-joining factor, we identify here histone H1 as an additional putative NHEJ factor. H1 strongly enhances DNA-end joining and shifts the product spectrum from circles to multimers. While H1 enhances the DNA-end-joining activities of both DNA Ligase IV and DNA Ligase III, the effect on ligase III is significantly stronger. Histone H1 also enhances the activity of PARP-1. Since histone H1 has been shown to counteract D-NHEJ, these observations and the known functions of the protein identify it as a putative alignment factor operating preferentially within B-NHEJ.

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The effect of histone H1 on DNA-end joining depends upon the amount and the length of the DNA substrate. (A) Titration of the effect of histone H1 on DNA-end joining for reactions assembled with 10 ng DNA Ligase IIIβ and different amounts of Sal I digested substrate DNA as indicated. Other conditions as in Figure 2C. (B) DNA-end joining in reactions assembled with 20 ng DNA Ligase IIIβ and 30 ng substrate DNA of different lengths as indicated. DNA fragments of 497 and 943 bp were prepared by digesting pUC19 with Alw44I. Other conditions as in Figure 2C. Alw44I recognizes the sequence 5′-G/TGCAC-3′ and generates ends with 3′ 4-bp extensions. (C) The choice of solvent modulates the activity of H1.2 in DNA-end joining. The experiments described earlier were carried out with H1.2 dissolved in reaction buffer (see ‘Materials and methods’). Titration of end-joining reactions with H1.2 dissolved in water or reaction buffer and tested in the presence of 10 ng DNA Ligase IIIβ. Other details as in Figure 2C. Note the shift in the maximum of DNA-end joining from 80 to 20 ng.
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Figure 3: The effect of histone H1 on DNA-end joining depends upon the amount and the length of the DNA substrate. (A) Titration of the effect of histone H1 on DNA-end joining for reactions assembled with 10 ng DNA Ligase IIIβ and different amounts of Sal I digested substrate DNA as indicated. Other conditions as in Figure 2C. (B) DNA-end joining in reactions assembled with 20 ng DNA Ligase IIIβ and 30 ng substrate DNA of different lengths as indicated. DNA fragments of 497 and 943 bp were prepared by digesting pUC19 with Alw44I. Other conditions as in Figure 2C. Alw44I recognizes the sequence 5′-G/TGCAC-3′ and generates ends with 3′ 4-bp extensions. (C) The choice of solvent modulates the activity of H1.2 in DNA-end joining. The experiments described earlier were carried out with H1.2 dissolved in reaction buffer (see ‘Materials and methods’). Titration of end-joining reactions with H1.2 dissolved in water or reaction buffer and tested in the presence of 10 ng DNA Ligase IIIβ. Other details as in Figure 2C. Note the shift in the maximum of DNA-end joining from 80 to 20 ng.

Mentions: The dramatic increase in multimers among the products of the reactions assembled with H1.2 suggests protein–DNA interactions that fundamentally alter substrate topology. Therefore, we investigated in greater detail the stoichiometry of H1.2 with reference to the substrate DNA. Figure 3A shows the effect of H1.2 on end-joining reactions assembled with 10 ng purified ligase III and different amounts of Sal I-linearized substrate DNA. While the end-joining maximum is achieved at 40 ng H1.2 when using 25 ng substrate, 80 ng is required in reactions assembled with 50 ng substrate. Further increase of substrate amount to 100 ng does not shift the maximum of end joining but reduces the inhibition typically observed at higher H1.2 concentrations. Assuming an even binding of H1 throughout the DNA molecule and considering the relative abundance in the reaction of substrate DNA and H1.2 protein, we estimate that at the end joining maximum there will be one H1 molecule bound for every 13 bp, if all supplied protein is active to bind DNA.Figure 3.


Histone H1 functions as a stimulatory factor in backup pathways of NHEJ.

Rosidi B, Wang M, Wu W, Sharma A, Wang H, Iliakis G - Nucleic Acids Res. (2008)

The effect of histone H1 on DNA-end joining depends upon the amount and the length of the DNA substrate. (A) Titration of the effect of histone H1 on DNA-end joining for reactions assembled with 10 ng DNA Ligase IIIβ and different amounts of Sal I digested substrate DNA as indicated. Other conditions as in Figure 2C. (B) DNA-end joining in reactions assembled with 20 ng DNA Ligase IIIβ and 30 ng substrate DNA of different lengths as indicated. DNA fragments of 497 and 943 bp were prepared by digesting pUC19 with Alw44I. Other conditions as in Figure 2C. Alw44I recognizes the sequence 5′-G/TGCAC-3′ and generates ends with 3′ 4-bp extensions. (C) The choice of solvent modulates the activity of H1.2 in DNA-end joining. The experiments described earlier were carried out with H1.2 dissolved in reaction buffer (see ‘Materials and methods’). Titration of end-joining reactions with H1.2 dissolved in water or reaction buffer and tested in the presence of 10 ng DNA Ligase IIIβ. Other details as in Figure 2C. Note the shift in the maximum of DNA-end joining from 80 to 20 ng.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 3: The effect of histone H1 on DNA-end joining depends upon the amount and the length of the DNA substrate. (A) Titration of the effect of histone H1 on DNA-end joining for reactions assembled with 10 ng DNA Ligase IIIβ and different amounts of Sal I digested substrate DNA as indicated. Other conditions as in Figure 2C. (B) DNA-end joining in reactions assembled with 20 ng DNA Ligase IIIβ and 30 ng substrate DNA of different lengths as indicated. DNA fragments of 497 and 943 bp were prepared by digesting pUC19 with Alw44I. Other conditions as in Figure 2C. Alw44I recognizes the sequence 5′-G/TGCAC-3′ and generates ends with 3′ 4-bp extensions. (C) The choice of solvent modulates the activity of H1.2 in DNA-end joining. The experiments described earlier were carried out with H1.2 dissolved in reaction buffer (see ‘Materials and methods’). Titration of end-joining reactions with H1.2 dissolved in water or reaction buffer and tested in the presence of 10 ng DNA Ligase IIIβ. Other details as in Figure 2C. Note the shift in the maximum of DNA-end joining from 80 to 20 ng.
Mentions: The dramatic increase in multimers among the products of the reactions assembled with H1.2 suggests protein–DNA interactions that fundamentally alter substrate topology. Therefore, we investigated in greater detail the stoichiometry of H1.2 with reference to the substrate DNA. Figure 3A shows the effect of H1.2 on end-joining reactions assembled with 10 ng purified ligase III and different amounts of Sal I-linearized substrate DNA. While the end-joining maximum is achieved at 40 ng H1.2 when using 25 ng substrate, 80 ng is required in reactions assembled with 50 ng substrate. Further increase of substrate amount to 100 ng does not shift the maximum of end joining but reduces the inhibition typically observed at higher H1.2 concentrations. Assuming an even binding of H1 throughout the DNA molecule and considering the relative abundance in the reaction of substrate DNA and H1.2 protein, we estimate that at the end joining maximum there will be one H1 molecule bound for every 13 bp, if all supplied protein is active to bind DNA.Figure 3.

Bottom Line: While H1 enhances the DNA-end-joining activities of both DNA Ligase IV and DNA Ligase III, the effect on ligase III is significantly stronger.Histone H1 also enhances the activity of PARP-1.Since histone H1 has been shown to counteract D-NHEJ, these observations and the known functions of the protein identify it as a putative alignment factor operating preferentially within B-NHEJ.

View Article: PubMed Central - PubMed

Affiliation: University of Duisburg-Essen, Medical School, Institute of Medical Radiation Biology, 45122 Essen, Germany.

ABSTRACT
DNA double-strand breaks (DSBs) induced in the genome of higher eukaryotes by ionizing radiation (IR) are predominantly removed by two pathways of non-homologous end-joining (NHEJ) termed D-NHEJ and B-NHEJ. While D-NHEJ depends on the activities of the DNA-dependent protein kinase (DNA-PK) and DNA ligase IV/XRCC4/XLF, B-NHEJ utilizes, at least partly, DNA ligase III/XRCC1 and PARP-1. Using in vitro end-joining assays and protein fractionation protocols similar to those previously applied for the characterization of DNA ligase III as an end-joining factor, we identify here histone H1 as an additional putative NHEJ factor. H1 strongly enhances DNA-end joining and shifts the product spectrum from circles to multimers. While H1 enhances the DNA-end-joining activities of both DNA Ligase IV and DNA Ligase III, the effect on ligase III is significantly stronger. Histone H1 also enhances the activity of PARP-1. Since histone H1 has been shown to counteract D-NHEJ, these observations and the known functions of the protein identify it as a putative alignment factor operating preferentially within B-NHEJ.

Show MeSH
Related in: MedlinePlus