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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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Identification of H1 as a DNA-end-joining factor. (A) Outline of the nuclear extract fractionation scheme utilized. Extracts were first fractionated over a double-stranded DNA cellulose column followed by further fractionation of active fractions over a Mono S column. Shown are the general fractionation schemes, typical chromatograms for each column, as well as SDS–PAGE gels of peak fractions. (B) Effect of fraction IIID on DNA-end joining catalyzed by purified DNA Ligase IIIβ. Reactions were assembled with the indicated amounts of protein. Shown as control are reactions assembled without extract, or with crude nuclear extract. (C) Coomassie blue-stained SDS–PAGE gel (12%) of fractions 86 and 87 (IIID, 20 µl) from a fractionation over a Mono S column. The two prominent bands detected were excised and subjected to mass spectrometry analysis. (D) Sequest search of peptides characterized by LC/MS-MS from the bands shown in C led to the identification of histone H1 variants H1.4 and H1.5 in the upper band and histone H1.2, H1.4 and H1.5 (74) in the lower band (derived by Flicka sequence analysis). The graph shows the corresponding isoforms and the coverage achieved through the peptides analyzed (grey boxes; lines indicate the size of the covered area and the size of the protein isoform itself). (E) Western blot analysis of the indicated fractions with an anti-histone H1 antibody. Ten micrograms of HeLa nuclear extract (NE) and each 10 µl of fractions I/II and III, IIIB1 and IIIB2 as well as 2 µl of fraction IIID2 were separated on a 12% SDS–PAGE gel and blotted onto a nitrocellulose membrane.
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Figure 1: Identification of H1 as a DNA-end-joining factor. (A) Outline of the nuclear extract fractionation scheme utilized. Extracts were first fractionated over a double-stranded DNA cellulose column followed by further fractionation of active fractions over a Mono S column. Shown are the general fractionation schemes, typical chromatograms for each column, as well as SDS–PAGE gels of peak fractions. (B) Effect of fraction IIID on DNA-end joining catalyzed by purified DNA Ligase IIIβ. Reactions were assembled with the indicated amounts of protein. Shown as control are reactions assembled without extract, or with crude nuclear extract. (C) Coomassie blue-stained SDS–PAGE gel (12%) of fractions 86 and 87 (IIID, 20 µl) from a fractionation over a Mono S column. The two prominent bands detected were excised and subjected to mass spectrometry analysis. (D) Sequest search of peptides characterized by LC/MS-MS from the bands shown in C led to the identification of histone H1 variants H1.4 and H1.5 in the upper band and histone H1.2, H1.4 and H1.5 (74) in the lower band (derived by Flicka sequence analysis). The graph shows the corresponding isoforms and the coverage achieved through the peptides analyzed (grey boxes; lines indicate the size of the covered area and the size of the protein isoform itself). (E) Western blot analysis of the indicated fractions with an anti-histone H1 antibody. Ten micrograms of HeLa nuclear extract (NE) and each 10 µl of fractions I/II and III, IIIB1 and IIIB2 as well as 2 µl of fraction IIID2 were separated on a 12% SDS–PAGE gel and blotted onto a nitrocellulose membrane.

Mentions: We have previously shown (32) that the DNA-end-joining activity of HeLa nuclear extract can be fractionated according to the scheme outlined in Figure 1A. First, ds-DNA cellulose effectively separates the majority of end-joining activity in the 750 mM NaCl fraction (fraction III). Further fractionation on Mono S generates fraction IIIB2, which contains the majority of DNA-end-joining activity (Figure 1A). Fraction IIIB1 has only limited activity and fraction IIIC is inactive—either by itself or in combination with other fractions [Figure 1A and (32)]. Factors implicated in D-NHEJ, such as DNA ligase IV, DNA-PKcs and Ku are mainly found in IIIB1, whereas DNA Ligase III, PARP-1 and XRCC1 are mainly found in IIIB2 (32). Fraction IIID is inactive by itself, but increases significantly the end-joining activity of IIIB2 suggesting an important role in the reaction (32). Yet, the active factor in IIID remained uncharacterized.Figure 1.


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)

Identification of H1 as a DNA-end-joining factor. (A) Outline of the nuclear extract fractionation scheme utilized. Extracts were first fractionated over a double-stranded DNA cellulose column followed by further fractionation of active fractions over a Mono S column. Shown are the general fractionation schemes, typical chromatograms for each column, as well as SDS–PAGE gels of peak fractions. (B) Effect of fraction IIID on DNA-end joining catalyzed by purified DNA Ligase IIIβ. Reactions were assembled with the indicated amounts of protein. Shown as control are reactions assembled without extract, or with crude nuclear extract. (C) Coomassie blue-stained SDS–PAGE gel (12%) of fractions 86 and 87 (IIID, 20 µl) from a fractionation over a Mono S column. The two prominent bands detected were excised and subjected to mass spectrometry analysis. (D) Sequest search of peptides characterized by LC/MS-MS from the bands shown in C led to the identification of histone H1 variants H1.4 and H1.5 in the upper band and histone H1.2, H1.4 and H1.5 (74) in the lower band (derived by Flicka sequence analysis). The graph shows the corresponding isoforms and the coverage achieved through the peptides analyzed (grey boxes; lines indicate the size of the covered area and the size of the protein isoform itself). (E) Western blot analysis of the indicated fractions with an anti-histone H1 antibody. Ten micrograms of HeLa nuclear extract (NE) and each 10 µl of fractions I/II and III, IIIB1 and IIIB2 as well as 2 µl of fraction IIID2 were separated on a 12% SDS–PAGE gel and blotted onto a nitrocellulose membrane.
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Figure 1: Identification of H1 as a DNA-end-joining factor. (A) Outline of the nuclear extract fractionation scheme utilized. Extracts were first fractionated over a double-stranded DNA cellulose column followed by further fractionation of active fractions over a Mono S column. Shown are the general fractionation schemes, typical chromatograms for each column, as well as SDS–PAGE gels of peak fractions. (B) Effect of fraction IIID on DNA-end joining catalyzed by purified DNA Ligase IIIβ. Reactions were assembled with the indicated amounts of protein. Shown as control are reactions assembled without extract, or with crude nuclear extract. (C) Coomassie blue-stained SDS–PAGE gel (12%) of fractions 86 and 87 (IIID, 20 µl) from a fractionation over a Mono S column. The two prominent bands detected were excised and subjected to mass spectrometry analysis. (D) Sequest search of peptides characterized by LC/MS-MS from the bands shown in C led to the identification of histone H1 variants H1.4 and H1.5 in the upper band and histone H1.2, H1.4 and H1.5 (74) in the lower band (derived by Flicka sequence analysis). The graph shows the corresponding isoforms and the coverage achieved through the peptides analyzed (grey boxes; lines indicate the size of the covered area and the size of the protein isoform itself). (E) Western blot analysis of the indicated fractions with an anti-histone H1 antibody. Ten micrograms of HeLa nuclear extract (NE) and each 10 µl of fractions I/II and III, IIIB1 and IIIB2 as well as 2 µl of fraction IIID2 were separated on a 12% SDS–PAGE gel and blotted onto a nitrocellulose membrane.
Mentions: We have previously shown (32) that the DNA-end-joining activity of HeLa nuclear extract can be fractionated according to the scheme outlined in Figure 1A. First, ds-DNA cellulose effectively separates the majority of end-joining activity in the 750 mM NaCl fraction (fraction III). Further fractionation on Mono S generates fraction IIIB2, which contains the majority of DNA-end-joining activity (Figure 1A). Fraction IIIB1 has only limited activity and fraction IIIC is inactive—either by itself or in combination with other fractions [Figure 1A and (32)]. Factors implicated in D-NHEJ, such as DNA ligase IV, DNA-PKcs and Ku are mainly found in IIIB1, whereas DNA Ligase III, PARP-1 and XRCC1 are mainly found in IIIB2 (32). Fraction IIID is inactive by itself, but increases significantly the end-joining activity of IIIB2 suggesting an important role in the reaction (32). Yet, the active factor in IIID remained uncharacterized.Figure 1.

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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