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

Extracts of Xenopus eggs and of cultured human and hamster cells have the capacity to join nonhomologous DNA ends, and all do so with similar specificity. To examine the formation of repair complexes on DNA under conditions of end joining, end‐labeled fragments were incubated with the various extrac...

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Veröffentlicht in:Environmental and molecular mutagenesis 2003, Vol.42 (4), p.279-287
Hauptverfasser: Lee, Jae Wan, Inamdar, Kedar V., Hannah, Michele F., Lees-Miller, Susan P., Povirk, Lawrence F.
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container_issue 4
container_start_page 279
container_title Environmental and molecular mutagenesis
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creator Lee, Jae Wan
Inamdar, Kedar V.
Hannah, Michele F.
Lees-Miller, Susan P.
Povirk, Lawrence F.
description Extracts of Xenopus eggs and of cultured human and hamster cells have the capacity to join nonhomologous DNA ends, and all do so with similar specificity. To examine the formation of repair complexes on DNA under conditions of end joining, end‐labeled fragments were incubated with the various extracts and then subjected to DNase‐I footprinting. Human and Xenopus extracts produced footprints virtually identical to that of purified DNA‐dependent protein kinase holoenzyme (Ku plus DNA‐PKcs), with protection of the terminal 28 bp. Extracts of hamster cells were more variable, but usually produced a 16‐bp footprint, similar to that of Ku alone. In all cases a 28‐bp holoenzyme‐like footprint was associated with wortmannin‐sensitive end joining, minimal 3′‐5′ exonucleolytic resection, and a predominance of accurate end‐joining products. To determine whether the short segments of DNA occupied by Ku and DNA‐PK were sufficient to support end joining, Y‐shaped substrates were constructed in which only one arm was available for end joining. A Y substrate with a 31‐bp arm bearing a partially cohesive 3′ overhang was accurately joined by a Xenopus egg extract, whereas a substrate with a 21‐bp arm was not. Surprisingly, a human cell extract did not join the Y substrates at all. The results suggest that differences in wortmannin sensitivity and in the distribution of in vitro end‐joining products may be attributable to the variations in the levels of DNA‐PKcs in the extracts. In addition, end joining in human extracts appears to involve interactions with significantly longer segments of DNA than the ∼28 bp occupied by DNA‐PK. Environ. Mol. Mutagen. 42:279–287, 2003. © 2003 Wiley‐Liss, Inc.
doi_str_mv 10.1002/em.10197
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To examine the formation of repair complexes on DNA under conditions of end joining, end‐labeled fragments were incubated with the various extracts and then subjected to DNase‐I footprinting. Human and Xenopus extracts produced footprints virtually identical to that of purified DNA‐dependent protein kinase holoenzyme (Ku plus DNA‐PKcs), with protection of the terminal 28 bp. Extracts of hamster cells were more variable, but usually produced a 16‐bp footprint, similar to that of Ku alone. In all cases a 28‐bp holoenzyme‐like footprint was associated with wortmannin‐sensitive end joining, minimal 3′‐5′ exonucleolytic resection, and a predominance of accurate end‐joining products. To determine whether the short segments of DNA occupied by Ku and DNA‐PK were sufficient to support end joining, Y‐shaped substrates were constructed in which only one arm was available for end joining. A Y substrate with a 31‐bp arm bearing a partially cohesive 3′ overhang was accurately joined by a Xenopus egg extract, whereas a substrate with a 21‐bp arm was not. Surprisingly, a human cell extract did not join the Y substrates at all. The results suggest that differences in wortmannin sensitivity and in the distribution of in vitro end‐joining products may be attributable to the variations in the levels of DNA‐PKcs in the extracts. In addition, end joining in human extracts appears to involve interactions with significantly longer segments of DNA than the ∼28 bp occupied by DNA‐PK. Environ. Mol. 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Mol. Mutagen</addtitle><date>2003</date><risdate>2003</risdate><volume>42</volume><issue>4</issue><spage>279</spage><epage>287</epage><pages>279-287</pages><issn>0893-6692</issn><eissn>1098-2280</eissn><coden>EMMUEG</coden><abstract>Extracts of Xenopus eggs and of cultured human and hamster cells have the capacity to join nonhomologous DNA ends, and all do so with similar specificity. To examine the formation of repair complexes on DNA under conditions of end joining, end‐labeled fragments were incubated with the various extracts and then subjected to DNase‐I footprinting. Human and Xenopus extracts produced footprints virtually identical to that of purified DNA‐dependent protein kinase holoenzyme (Ku plus DNA‐PKcs), with protection of the terminal 28 bp. Extracts of hamster cells were more variable, but usually produced a 16‐bp footprint, similar to that of Ku alone. 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subjects Androstadienes - pharmacology
Animals
Antigens, Nuclear - chemistry
Biological and medical sciences
Cell Extracts
Chemical mutagenesis
CHO Cells
Cricetinae
Deoxyribonuclease I - metabolism
DNA - chemistry
DNA Damage
DNA Helicases
DNA Repair
DNA-Activated Protein Kinase
DNA-Binding Proteins - chemistry
DNA-dependent protein kinase
DNase footprinting
Enzyme Inhibitors - pharmacology
Humans
In Vitro Techniques
Ku antigen
Ku Autoantigen
Medical sciences
nonhomologous end joining
Nuclear Proteins
Plasmids - metabolism
Protein-Serine-Threonine Kinases - metabolism
Toxicology
Xenopus
title DNA end sequestration by DNA-dependent protein kinase and end joining of sterically constrained substrates in whole-cell extracts
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