Reconstitution of the mammalian DNA double-strand break end-joining reaction reveals a requirement for an Mre11/Rad50/NBS1-containing fraction

被引:105
作者
Huang, JR [1 ]
Dynan, WS [1 ]
机构
[1] Med Coll Georgia, Inst Mol Med & Genet, Program Gene Regulat, Augusta, GA 30912 USA
关键词
D O I
10.1093/nar/30.3.667
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The non-homologous end-joining pathway promotes direct enzymatic rejoining of DNA double-strand breaks (DSBs) and is an important determinant of genome stability in eukaryotic cells. Although previous work has shown that this pathway requires Ku, DNA-PKcs and the DNA ligase IV/XRCC4 complex, we found that these proteins alone did not promote efficient joining of cohesive-ended DNA fragments in a cell-free assay. To identify factors that were missing from the reaction, we screened fractions from HeLa cell extracts for the ability to stimulate the joining of cohesive DNA ends in a complementation assay containing other known proteins required for DNA DSB repair. We identified a factor that restored end-joining activity to the level observed in crude nuclear extracts. Factor activity copurified with Rad50, Mre11 and NBS1, three proteins that have previously been implicated in DSB repair by genetic and cytologic evidence. Factor activity was inhibited by anti-Mre11 antibody. The reconstituted system remained fully dependent on DNL IV/XRCC4 and at least partially dependent on Ku, but the requirement for DNA-PKcs was progressively lost as other components were purified. Results support a model where DNA-PKcs acts early in the DSB repair pathway to regulate progression of the reaction, and where Mre11, Rad50 and NBS1 play a key role in aligning DNA ends in a synaptic complex immediately prior to ligation.
引用
收藏
页码:667 / 674
页数:8
相关论文
共 75 条
[51]   The 3′ to 5′ exonuclease activity of Mre11 facilitates repair of DNA double-strand breaks [J].
Paull, TT ;
Gellert, M .
MOLECULAR CELL, 1998, 1 (07) :969-979
[52]   A mechanistic basis for Mre11-directed DNA joining at microhomologies [J].
Paull, TT ;
Gellert, M .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2000, 97 (12) :6409-6414
[53]   LOSS OF THE CATALYTIC SUBUNIT OF THE DNA-DEPENDENT PROTEIN-KINASE IN DNA DOUBLE-STRAND-BREAK-REPAIR MUTANT MAMMALIAN-CELLS [J].
PETERSON, SR ;
KURIMASA, A ;
OSHIMURA, M ;
DYNAN, WS ;
BRADBURY, EM ;
CHEN, DJ .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1995, 92 (08) :3171-3174
[54]   JOINING OF NONHOMOLOGOUS DNA DOUBLE STRAND BREAKS INVITRO [J].
PFEIFFER, P ;
VIELMETTER, W .
NUCLEIC ACIDS RESEARCH, 1988, 16 (03) :907-924
[55]   Ku protein stimulates DNA end joining by mammalian DNA ligases: a direct role for Ku in repair of DNA double-strand breaks [J].
Ramsden, DA ;
Gellert, M .
EMBO JOURNAL, 1998, 17 (02) :609-614
[56]   Megabase chromatin domains involved in DNA double-strand breaks in vivo [J].
Rogakou, EP ;
Boon, C ;
Redon, C ;
Bonner, WM .
JOURNAL OF CELL BIOLOGY, 1999, 146 (05) :905-915
[57]   Normal telomere length and chromosomal end capping in poly(ADP-ribose) polymerase-deficient mice and primary cells despite increased chromosomal instability [J].
Samper, E ;
Goytisolo, FA ;
Ménissier-de Murcia, J ;
González-Suárez, E ;
Cigudosa, JC ;
de Murcia, G ;
Blasco, MA .
JOURNAL OF CELL BIOLOGY, 2001, 154 (01) :49-60
[58]   A newly identified DNA ligase of Saccharomyces cerevisiae involved in RAD52-independent repair of DNA double-strand breaks [J].
Schar, P ;
Herrmann, G ;
Daly, G ;
Lindahl, T .
GENES & DEVELOPMENT, 1997, 11 (15) :1912-1924
[59]   p53 Binding protein 1 (53BP1) is an early participant in the cellular response to DNA double-strand breaks [J].
Schultz, LB ;
Chehab, NH ;
Malikzay, A ;
Halazonetis, TD .
JOURNAL OF CELL BIOLOGY, 2000, 151 (07) :1381-1390
[60]   Dynamic changes of BRCA1 subnuclear location and phosphorylation state are initiated by DNA damage [J].
Scully, R ;
Chen, JJ ;
Ochs, RL ;
Keegan, K ;
Hoekstra, M ;
Feunteun, J ;
Livingston, DM .
CELL, 1997, 90 (03) :425-435