Phosphorylation of linker histones by DNA-dependent protein kinase is required for DNA ligase IV-dependent ligation in the presence of histone H1

Kysela, Boris; Chovanec, Miroslav; Jeggo, Penny A.

doi:10.1073/pnas.0401179102

Cited by 49 publications

(43 citation statements)

References 46 publications

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“…Ku Displaces Histone H1 from DNA Ends-Binding of protein to DNA fragments has been shown in vitro to inhibit the ability of XRCC4-Ligase IV to join DNA ends (26,27). Interestingly, Ku and DNA-PKcs are required to relieve the specific inhibition caused by the presence of the linker histone H1 (27), suggesting Ku may play roles in recognizing H1-occluded DNA ends and making these ends accessible to DNA-PKcs and XRCC4-ligase IV.…”

Section: Resultsmentioning

confidence: 99%

Loading of the Nonhomologous End Joining Factor, Ku, on Protein-occluded DNA Ends

Roberts

Ramsden

2007

Journal of Biological Chemistry

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The nonhomologous end joining pathway for DNA double strand break repair requires Ku to bind DNA ends and subsequently recruit other nonhomologous end joining factors, including the DNA-dependent protein kinase catalytic subunit and the XRCC4-Ligase IV complex, to the break site. Ku loads at a break by threading the DNA ends through a circular channel in its structure. This binding mechanism explains both the high specificity of Ku for ends and its ability to translocate along DNA once loaded. However, DNA in cells is typically coated with other proteins (e.g. histones), which might be expected to block the ability of Ku to load in this manner. Here we address how the nature of a protein obstruction dictates how Ku interacts with a DNA end. Ku is unable to access the ends within an important intermediate in V(D)J recombination (a complex of RAG proteins bound to cleaved recombination targeting signals), but Ku readily displaces the linker histone, H1, from DNA. Ku also retains physiological affinity for nucleosome-associated ends. Loading onto nucleosome-associated ends still occurs by threading the end through its channel, but rather than displacing the nucleosome, Ku peels as much as 50 bp of DNA away from the histone octamer surface. We suggest a model where Ku utilizes an unusual characteristic of its three-dimensional structure to recognize certain protein-occluded ends without the extensive remodeling of chromatin structure required by other DNA repair pathways. DNA double strand breaks (DSBs)2 pose a threat to genomic integrity. Nonhomologous end joining (NHEJ) is a major pathway for repair of exogenously introduced DSBs in mammals and is the only efficient way to repair DSB intermediates in V(D)J recombination (1). NHEJ requires the DNA binding heterodimer Ku to first recognize broken DNA ends (2-5) and subsequently recruit the additional NHEJ factors necessary to complete repair (e.g. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) (6), the XRCC4-LigaseIV complex (7,8), and polymerases (9, 10)). Consequently, deficiency in Ku cripples NHEJ and leads to severe immunodeficiency (11, 12) and cellular sensitivity to agents that cause DSBs (e.g. ionizing radiation) (13,14). The ability of Ku to recognize DNA ends requires the end to be inserted through a channel in its structure (5, 15, 16). This manner of binding makes Ku highly specific for DNA ends but requires that the ends must be accessible through 360°. In cells, DNA is generally coated with proteins (e.g. histones and other chromatin-associated proteins) that might be expected to block the ability of Ku to load on DNA ends and thus impair NHEJ.Here we have shown that Ku displays a variety of responses to protein obstructions at DNA ends. Ku is unable to bind a class of RAG (recombination activating gene) protein-bound DNA ends generated during V(D)J recombination. In contrast, Ku can displace certain proteins (e.g. histone H1) from DNA and retains the ability to load on nucleosome-associated ends by peeling up to 50 bp of DNA away from the ...

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Section: Resultsmentioning

confidence: 99%

Loading of the Nonhomologous End Joining Factor, Ku, on Protein-occluded DNA Ends

Roberts

Ramsden

2007

Journal of Biological Chemistry

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“…For example, nucleosomes containing both H3.3 and H2AZ are highly unstable (27,28). Likewise, acetylation of the amino-terminal tails of the core histones decreases nucleosome stability, as does certain phosphorylations of linker histone H1 and core H3 (16,23,32,41,44,72). Histone variants and posttranslational modifications also correlate with transcriptional activity.…”

Section: Discussionmentioning

confidence: 99%

Herpes Simplex Virus 1 DNA Is in Unstable Nucleosomes throughout the Lytic Infection Cycle, and the Instability of the Nucleosomes Is Independent of DNA Replication

Lacasse

Schang

2012

J Virol

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Herpes simplex virus 1 (HSV-1) DNA is chromatinized during latency and consequently regularly digested by micrococcal nuclease (MCN) to nucleosome-size fragments. In contrast, MCN digests HSV-1 DNA in lytically infected cells to mostly heterogeneous sizes. Yet HSV-1 DNA coimmunoprecipitates with histones during lytic infections. We have shown that at 5 h postinfection, most nuclear HSV-1 DNA is in particularly unstable nucleoprotein complexes and consequently is more accessible to MCN than DNA in cellular chromatin. HSV-1 DNA was quantitatively recovered at this time in complexes with the biophysical properties of mono-to polynucleosomes following a modified MCN digestion developed to detect potential unstable intermediates. We proposed that most HSV-1 DNA is in unstable nucleosome-like complexes during lytic infections. Physiologically, nucleosome assembly typically associates with DNA replication, although DNA replication transiently disrupts nucleosomes. It therefore remained unclear whether the instability of the HSV-1 nucleoprotein complexes was related to the ongoing viral DNA replication. Here we tested whether HSV-1 DNA is in unstable nucleosome-like complexes before, during, or after the peak of viral DNA replication or when HSV-1 DNA replication is inhibited. HSV-1 DNA was quantitatively recovered in complexes fractionating as mono-to polynucleosomes from nuclei harvested at 2, 5, 7, or 9 h after infection, even if viral DNA replication was inhibited. Therefore, most HSV-1 DNA is in unstable nucleosome-like complexes throughout the lytic replication cycle, and the instability of these complexes is surprisingly independent of HSV-1 DNA replication. The specific accessibility of nuclear HSV-1 DNA, however, varied at different times after infection. E ukaryotic DNA is packaged into nucleoprotein complexes known as chromatin. The basic unit of chromatin is the nucleosome, 146 bp of DNA wrapped 1.75 turns around a histone octamer composed of two copies of each histone 2A (H2A), H2B, H3, and H4 (33). Linker histone H1 binds at the core nucleosome entry and exit points, stabilizing the core nucleosome, and to linker DNA in between nucleosomes. H1 also promotes further compaction of chromatin into higher-order structures (23,50,51). The chromatin structure is classically probed with nucleases (61). The endonuclease micrococcal nuclease (MCN), for example, preferentially cleaves linker DNA between nucleosomes (2, 19). Mild MCN digestions first cleave linker DNA sparsely, releasing polynucleosomes, which are eventually cleaved to mononucleosomes. Therefore, MCN digestion of regularly chromatinized DNA results in protection of polyand mononucleosome-size DNA fragments. Under more stringent digestion conditions, MCN eventually degrades even the DNA in mononucleosomes.MCN has also been used to characterize specialized chromatin structures and intrinsically unstable nucleosomes. Centromeres, for example, are specialized chromatin structures responsible for the equal segregation of chromosomes at mitosis. They contai...

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“…4) may also have an important contribution to these processes (59). Indeed, DNA-PK-mediated phosphorylation of H1 has been shown to decrease its affinity within the DNA repair context (59).…”

Section: Discussionmentioning

confidence: 99%

Phosphorylation of Histone H2A.X by DNA-dependent Protein Kinase Is Not Affected by Core Histone Acetylation, but It Alters Nucleosome Stability and Histone H1 Binding

Lee

et al. 2010

Journal of Biological Chemistry

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Phosphorylation of the C-terminal end of histone H2A.X is the most characterized histone post-translational modification in DNA double-stranded breaks (DSB). DNA-dependent protein kinase (DNA-PK) is one of the three phosphatidylinositol 3 kinase-like family of kinase members that is known to phosphorylate histone H2A.X during DNA DSB repair. There is a growing body of evidence supporting a role for histone acetylation in DNA DSB repair, but the mechanism or the causative relation remains largely unknown. Using bacterially expressed recombinant mutants and stably and transiently transfected cell lines, we find that DNA-PK can phosphorylate Thr-136 in addition to Ser-139 both in vitro and in vivo. Furthermore, the phosphorylation reaction is not inhibited by the presence of H1, which in itself is a substrate of the reaction. We also show that, in contrast to previous reports, the ability of the enzyme to phosphorylate these residues is not affected by the extent of acetylation of the core histones. In vitro assembled nucleosomes and HeLa S3 native oligonucleosomes consisting of non-acetylated and acetylated histones are equally phosphorylated by DNA-PK. We demonstrate that the apparent differences in the extent of phosphorylation previously observed can be accounted for by the differential chromatin solubility under the MgCl 2 concentrations required for the phosphorylation reaction in vitro. Finally, we show that although H2A.X does not affect nucleosome conformation, it has a de-stabilizing effect that is enhanced by the DNA-PK-mediated phosphorylation and results in an impaired histone H1 binding. DNA double-stranded break (DSB)3 repair is an important cellular process that is critical for the maintenance of genome integrity. If improperly repaired, DSBs can have detrimental consequences leading to cancer via chromosomal aberrations such as chromosomal breaks, translocation, and aneuploidy (1).In the eukaryotic cell the repair mechanism requires the participation of chromatin components. One of the chromatin hallmarks involved is the phosphorylation of histone H2A.X at its highly conserved C-terminal SQE motif (Fig. 1A) (2-4). This phosphorylated form of H2A.X is commonly denoted as ␥-H2A.X (2). It is well known that histone H2A.X is phosphorylated ubiquitously at DNA DSB regardless of the source of the damage and of the pathway used in its repair: homologous recombination or non-homologous-end joining (NHEJ). The enzymes responsible for the DSB-induced H2A.X phosphorylation are all members of the PIKK family, such as the DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) (4, 5). The DNA-PK catalytic subunit has been recently shown to play a dominant role in regulating the DNA damage-mediated phosphorylation of H2A.X (6).In the yeast H2A.X ortholog, Thr-126 and Ser-129 can be both phosphorylated during DSB in a way that appears to depend on the repair pathway. It has been shown that Thr-126 is important for homologous recombination but dispensable for NHEJ ...

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Phosphorylation of linker histones by DNA-dependent protein kinase is required for DNA ligase IV-dependent ligation in the presence of histone H1

Cited by 49 publications

References 46 publications

Loading of the Nonhomologous End Joining Factor, Ku, on Protein-occluded DNA Ends

Loading of the Nonhomologous End Joining Factor, Ku, on Protein-occluded DNA Ends

Herpes Simplex Virus 1 DNA Is in Unstable Nucleosomes throughout the Lytic Infection Cycle, and the Instability of the Nucleosomes Is Independent of DNA Replication

Phosphorylation of Histone H2A.X by DNA-dependent Protein Kinase Is Not Affected by Core Histone Acetylation, but It Alters Nucleosome Stability and Histone H1 Binding

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