Possible involvement of RecQL4 in the repair of double-strand DNA breaks in Xenopus egg extracts.
Mutations in RecQL4 are a causative factor in Rothmund-Thomson syndrome, a human autosomal recessive disorder characterized by premature aging. To study the role of RecQL4, we employed a cell-free experimental system consisting of Xenopus egg extracts. RecQL4 loading onto chromatin was observed regardless of the presence or absence of EcoRI. However, in the absence of EcoRI, RecQL4 loading was suppressed by geminin, an inhibitor of pre-replicative complex formation, while in the presence of EcoRI, it was not affected. These results suggest that under the former condition, RecQL4-loading depended on DNA replication, while under the latter, the interaction occurred in response to double-stranded DNA breaks (DSBs) induced by EcoRI. DSB-induced RecQL4 loading depended on the function of the ataxia-telangiectasia mutated protein, DNA-dependent protein kinase (DNA-PK), and replication protein A, while there were only minor changes in DNA replication-associated RecQL4 loading upon suppression of these proteins. Furthermore, analyses using a chromatin-immunoprecipitation assay and quantification of gammaH2AX after induction of DSBs suggested that RecQL4 is loaded adjacent to Ku heterodimer-binding sites on damaged chromatin, and functions in the repair of DSBs.
Kumata
Yuji
Y
Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan.
Tada
Shusuke
S
Yamanada
Yumie
Y
Tsuyama
Takashi
T
Kobayashi
Takayuki
T
Dong
Yu-Peng
YP
Ikegami
Kyoko
K
Murofushi
Hiromu
H
Seki
Masayuki
M
Enomoto
Takemi
T
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002457:Cell Extracts; D002843:Chromatin / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects; D004260:DNA Repair / Q000187:drug effects; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D006801:Humans; D010063:Ovum / Q000187:drug effects / Q000201:enzymology*; D021381:Protein Transport / Q000187:drug effects; D051135:Rad51 Recombinase / Q000378:metabolism; D053484:RecQ Helicases / Q000378:metabolism*; D051716:Replication Protein A / Q000172:deficiency; D013997:Time Factors; D000077191:Wortmannin; D014981:Xenopus / Q000378:metabolism*; D029867:Xenopus Proteins / Q000378:metabolism
D000730:Androstadienes; D002457:Cell Extracts; D002843:Chromatin; C468783:H2AX protein, human; D006657:Histones; D051716:Replication Protein A; D029867:Xenopus Proteins; D002110:Caffeine; C495152:RAD51 protein, Xenopus; D051135:Rad51 Recombinase; D015246:Deoxyribonuclease EcoRI; D053484:RecQ Helicases; D000077191:Wortmannin