DNA end sequestration by DNA‐dependent protein kinase and end joining of sterically constrained substrates in whole‐cell extracts

Lee, Jae Wan; Inamdar, Kedar; Hannah, Michele F.; Lees‐Miller, Susan P.; Povirk, Lawrence F.

doi:10.1002/em.10197

Cited by 6 publications

(4 citation statements)

References 39 publications

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“…This result indicates that all the protein complexes, irrespective of their apparent mass, did assemble onto the very end of the oligonucleotides. This observation is compatible with the 16 bp minimal length of duplex DNA required to assemble protein complexes ( 29 , 30 ).…”

Section: Resultssupporting

confidence: 81%

The human DNA ends proteome uncovers an unexpected entanglement of functional pathways

et al. 2016

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DNA ends get exposed in cells upon either normal or dysfunctional cellular processes or molecular events. Telomeres need to be protected by the shelterin complex to avoid junctions occurring between chromosomes while failing topoisomerases or clustered DNA damage processing may produce double-strand breaks, thus requiring swift repair to avoid cell death. The rigorous study of the great many proteins involved in the maintenance of DNA integrity is a challenging task because of the innumerous unspecific electrostatic and/or hydrophobic DNA—protein interactions that arise due to the chemical nature of DNA. We devised a technique that discriminates the proteins recruited specifically at DNA ends from those that bind to DNA because of a generic affinity for the double helix. Our study shows that the DNA ends proteome comprises proteins of an unexpectedly wide functional spectrum, ranging from DNA repair to ribosome biogenesis and cytoskeleton, including novel proteins of undocumented function. A global mapping of the identified proteome on published DNA repair protein networks demonstrated the excellent specificity and functional coverage of our purification technique. Finally, the native nucleoproteic complexes that assembled specifically onto DNA ends were shown to be endowed with a highly efficient DNA repair activity.

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Section: Resultssupporting

confidence: 81%

The human DNA ends proteome uncovers an unexpected entanglement of functional pathways

et al. 2016

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“…The machinery of classical NHEJ, incorporating DNA-PK, XLF, X4L4 and polymerases λ and μ, has a remarkable capacity to anneal, patch and ligate DNA ends having minimal and imperfect complementarity [35]. Nevertheless, blocked termini and adjacent DNA base damage can constitute a significant barrier to end joining [17,38–40], and although a variety of enzymes are available for resolving these terminal modifications, ends sequestered by end joining proteins may not be accessible to those enzymes [41]. Moreover, even if accessible, heavily damaged ends with multiple modifications may not be recognized as substrates by repair enzymes.…”

Section: Discussionmentioning

confidence: 99%

Trimming of damaged 3′ overhangs of DNA double-strand breaks by the Metnase and Artemis endonucleases

et al. 2013

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Both Metnase and Artemis possess endonuclease activities that trim 3′ overhangs of duplex DNA. To assess the potential of these enzymes for facilitating resolution of damaged ends during double-strand break rejoining, substrates bearing a variety of normal and structurally modified 3′ overhangs were constructed, and treated either with Metnase or with Artemis plus DNA-dependent protein kinase (DNA-PK). Unlike Artemis, which trims long overhangs to 4–5 bases, cleavage by Metnase was more evenly distributed over the length of the overhang, but with significant sequence dependence. In many substrates, Metnase also induced marked cleavage in the double-stranded region within a few bases of the overhang. Like Artemis, Metnase efficiently trimmed overhangs terminated in 3′-phosphoglycolates (PGs), and in some cases the presence of 3′-PG stimulated cleavage and altered its specificity. The nonplanar base thymine glycol in a 3′ overhang severely inhibited cleavage by Metnase in the vicinity of the modified base, while Artemis was less affected. Nevertheless, thymine glycol moieties could be removed by Metnase- or Artemis-mediated cleavage at sites farther from the terminus than the lesion itself. In in vitro end-joining systems based on human cell extracts, addition of Artemis, but not Metnase, effected robust trimming of an unligatable 3′-PG overhang, resulting in a dramatic stimulation of ligase IV- and XLF-dependent end joining. Thus, while both Metnase and Artemis are biochemically capable of resolving a variety of damaged DNA ends for the repair of complex double-strand breaks, Artemis appears to act more efficiently in the context of other nonhomologous end joining proteins.

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“…As footprinting studies indicate that Ku plus DNA-PKcs protects ϳ28 bp at a DNA end (18), the apparent requirement for at least ϳ20 bp of duplex DNA for proper binding and alignment of the nuclease complex is not surprising.…”

Section: Trimming Of Long 3ј-pg-terminated Overhangs By Artemis-mentioning

confidence: 99%

Processing of 3′-Phosphoglycolate-terminated DNA Double Strand Breaks by Artemis Nuclease

Povirk

Zhou

et al. 2007

Journal of Biological Chemistry

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The Artemis nuclease is required for V(D)J recombination and for repair of an as yet undefined subset of radiation-induced DNA double strand breaks. To assess the possibility that Artemis acts on oxidatively modified double strand break termini, its activity toward model DNA substrates, bearing either 3-hydroxyl or 3-phosphoglycolate moieties, was examined. A 3-phosphoglycolate had little effect on Artemis-mediated trimming of long 3 overhangs (>9 nucleotides), which were efficiently trimmed to 4 -5 nucleotides. However, 3-phosphoglycolates on overhangs of 4 -5 bases promoted Artemis-mediated removal of a single 3-terminal nucleotide, while at least 2 nucleotides were trimmed from identical hydroxyl-terminated substrates. Artemis also efficiently removed a single nucleotide from a phosphoglycolate-terminated 3-base 3 overhang, while leaving an analogous hydroxyl-terminated overhang largely intact. Such removal was completely dependent on DNA-dependent protein kinase and ATP and was largely dependent on Ku, which markedly stimulated Artemis activity toward all 3 overhangs. Together, these data suggest that efficient Artemismediated cleavage of 3 overhangs requires a minimum of 2 nucleotides, or a nucleotide plus a phosphoglycolate, 3 to the cleavage site, as well as 2 unpaired nucleotides 5 to the cleavage site. Shorter 3-phosphoglycolate-terminated overhangs and blunt ends were also processed by Artemis but much more slowly. Consistent with a role for Artemis in repair of terminally blocked double strand breaks in vivo, human cells lacking Artemis exhibited hypersensitivity to x-rays, bleomycin, and neocarzinostatin, which all induce 3-phosphoglycolate-terminated double strand breaks.The Artemis genetic locus was identified by virtue of its association with a form of BϪ TϪ NKϩ severe combined immune deficiency (SCID) 2 in humans, designated RS-SCID (radiationsensitive SCID) (1) or SCIDA (Athabascan SCID) (2). The Artemis protein is a nuclease that is activated by DNA-dependent protein kinase (DNA-PK) and is required for the opening of hairpin ends formed during V(D)J recombination (3), thus accounting for the SCID phenotype associated with Artemis deficiency. SCIDA and RS-SCID fibroblasts are radiation-sensitive but fail to repair only a small fraction of radiation-induced DNA double strand breaks (DSBs) (4 -6). In vitro, activated Artemis removes 5Ј overhangs from DNA ends and shortens 3Ј overhangs (7), raising the possibility that during DSB repair in vivo, Artemis may trim overhangs that otherwise cannot be processed to give ligatable ends.About half of DNA breaks induced by ionizing radiation bear 3Ј-phosphoglycolate (3Ј-PG) termini in various contexts (7) that must be removed in order to allow gap filling by DNA polymerases and and ligation by DNA ligase IV (8). Although tyrosyl-DNA phosphodiesterase (TDP1) is the only identified enzyme capable of processing 3Ј-PGs on 3Ј overhangs (9), TDP1 mutant cells show only marginal radiosensitivity (10), suggesting the existence of an alternative pathway for processi...

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DNA end sequestration by DNA‐dependent protein kinase and end joining of sterically constrained substrates in whole‐cell extracts

Cited by 6 publications

References 39 publications

The human DNA ends proteome uncovers an unexpected entanglement of functional pathways

The human DNA ends proteome uncovers an unexpected entanglement of functional pathways

Trimming of damaged 3′ overhangs of DNA double-strand breaks by the Metnase and Artemis endonucleases

Processing of 3′-Phosphoglycolate-terminated DNA Double Strand Breaks by Artemis Nuclease

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