Distinct populations of human PCNA are required for initiation of chromosomal DNA replication and concurrent DNA repair

被引:22
作者
Szüts, D [1 ]
Christov, C [1 ]
Kitching, L [1 ]
Krude, T [1 ]
机构
[1] Univ Cambridge, Dept Zool, Cambridge CB2 3EJ, England
关键词
human chromosomal DNA replication; cell cycle; DNA damage signalling; in vitro system;
D O I
10.1016/j.yexcr.2005.09.009
中图分类号
R73 [肿瘤学];
学科分类号
100214 ;
摘要
The integrity of genomic DNA during the cell division cycle in eukaryotic cells is maintained by regulated chromosomal DNA replication and repair of damaged DNA. We have used fractionation and reconstitution experiments to purify essential factors for the initiation of human chromosomal DNA replication in late G1 phase template nuclei from human cells. Here, we report the identification of soluble PCNA as an essential initiation factor in this system. Recombinant histidine-tagged human PCNA can substitute for purified endogenous human PCNA to initiate human chromosomal DNA replication. It is recruited specifically to discrete DNA replication foci formed during initiation in vitro. The template nuclei also contain DNA breaks as result of the synchronisation procedure. A separate population of chromatin-bound PCNA is already present in these template nuclei at discrete DNA damage foci, co-localising with gamma-H2AX, RPA and Rad51. This DNA damage-associated PCNA population is marked by mono-ubiquitination, suggesting that it is involved in DNA repair. Importantly, the population of damage focus-associated PCNA is neither involved in, nor required for, the initiation of chromosomal DNA replication in the same nuclei. (c) 2005 Elsevier Inc. All rights reserved.
引用
收藏
页码:240 / 250
页数:11
相关论文
共 42 条
[1]   MOLECULAR-CLONING, STRUCTURE AND EXPRESSION OF THE YEAST PROLIFERATING CELL NUCLEAR ANTIGEN GENE [J].
BAUER, GA ;
BURGERS, PMJ .
NUCLEIC ACIDS RESEARCH, 1990, 18 (02) :261-265
[2]   Role of the human RAD51 protein in homologous recombination and double-stranded break repair [J].
Baumann, P ;
West, SC .
TRENDS IN BIOCHEMICAL SCIENCES, 1998, 23 (07) :247-251
[3]   DNA replication in eukaryotic cells [J].
Bell, SP ;
Dutta, A .
ANNUAL REVIEW OF BIOCHEMISTRY, 2002, 71 :333-374
[4]   Heterogeneity of eukaryotic replicons, replicon clusters, and replication foci [J].
Berezney, R ;
Dubey, DD ;
Huberman, JA .
CHROMOSOMA, 2000, 108 (08) :471-484
[5]   CDNAS ENCODING THE LARGE SUBUNIT OF HUMAN REPLICATION FACTOR-C [J].
BUNZ, F ;
KOBAYASHI, R ;
STILLMAN, B .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1993, 90 (23) :11014-11018
[6]   MCM2-7 complexes bind chromatin in a distributed pattern surrounding the origin recognition complex in Xenopus egg extracts [J].
Edwards, MC ;
Tutter, AV ;
Cvetic, C ;
Gilbert, CH ;
Prokhorova, TA ;
Walter, JC .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2002, 277 (36) :33049-33057
[7]   Fission yeast Cdc23/Mcm10 functions after prereplicative complex formation to promote Cdc45 chromatin binding [J].
Gregan, J ;
Lindner, K ;
Brimage, L ;
Franklin, R ;
Namdar, M ;
Hart, EA ;
Aves, SJ ;
Kearsey, SE .
MOLECULAR BIOLOGY OF THE CELL, 2003, 14 (09) :3876-3887
[8]   RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO [J].
Hoege, C ;
Pfander, B ;
Moldovan, GL ;
Pyrowolakis, G ;
Jentsch, S .
NATURE, 2002, 419 (6903) :135-141
[9]   Double-strand break repair in yeast requires both leading and lagging strand DNA polymerases [J].
Holmes, AM ;
Haber, JE .
CELL, 1999, 96 (03) :415-424
[10]   Replicon clusters are stable units of chromosome structure: Evidence that nuclear organization contributes to the efficient activation and propagation of S phase in human cells [J].
Jackson, DA ;
Pombo, A .
JOURNAL OF CELL BIOLOGY, 1998, 140 (06) :1285-1295