Increased origin activity in transformed versus normal cells: identification of novel protein players involved in DNA replication and cellular transformation.
Increased origin activity in transformed versus normal cells: identification of novel protein players involved in DNA replication and cellular transformation.
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DOI：
10.1093/nar/gkp1192
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发表时间：
2010-04
影响因子：
14.9
通讯作者：
Zannis-Hadjopoulos M
中科院分区：
文献类型：
--
作者：
Di Paola D;Rampakakis E;Chan MK;Arvanitis DN;Zannis-Hadjopoulos M
使用以前产生的复制起点库，我们确定了三个克隆，支持其各自的质粒在转化，但不是在正常细胞中的自主复制。通过原位染色体DNA复制测定评估它们的体内复制活性，揭示了对应于这些克隆的染色体位点与所有细胞系中的染色体复制起点一致，与正常细胞相比，转化细胞中的染色体复制起点的活性高2-3倍。通过染色质免疫沉淀测定，使用抗-ORC 2、-cdc 6和-cdt 1抗体，在转化细胞和正常细胞中这些起点处的复制前复合物（pre-RC）蛋白丰度的评价显示，它们在所有细胞系中被这些pre-RC蛋白结合，但与正常细胞相比，在转化细胞中观察到2-3倍的丰度。电泳迁移率变动分析（EMSA）进行的最有效的复制克隆，使用核提取物的转化和正常细胞，揭示了DNA复制复合物的存在下，在转化细胞，这是几乎检测不到的正常细胞。随后的超位移EMSA表明存在转化特异性复合物。对这些复合物的质谱分析揭示了参与DNA复制的潜在新蛋白质参与者，这些蛋白质似乎与细胞转化相关。
Using libraries of replication origins generated previously, we identified three clones that supported the autonomous replication of their respective plasmids in transformed, but not in normal cells. Assessment of their in vivo replication activity by in situ chromosomal DNA replication assays revealed that the chromosomal loci corresponding to these clones coincided with chromosomal replication origins in all cell lines, which were more active by 2–3-fold in the transformed by comparison to the normal cells. Evaluation of pre-replication complex (pre-RC) protein abundance at these origins in transformed and normal cells by chromatin immunoprecipitation assays, using anti-ORC2, -cdc6 and -cdt1 antibodies, showed that they were bound by these pre-RC proteins in all cell lines, but a 2–3-fold higher abundance was observed in the transformed by comparison to the normal cells. Electrophoretic mobility shift assays (EMSAs) performed on the most efficiently replicating clone, using nuclear extracts from the transformed and normal cells, revealed the presence of a DNA replication complex in transformed cells, which was barely detectable in normal cells. Subsequent supershift EMSAs suggested the presence of transformation-specific complexes. Mass spectrometric analysis of these complexes revealed potential new protein players involved in DNA replication that appear to correlate with cellular transformation.
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影响因子：
64.8
作者：
Bartkova, J;Horejsi, Z;Bartek, J
通讯作者：
Bartek, J
影响因子：
11.4
作者：
Abdurashidova, G;Danailov, MB;Falaschi, A
通讯作者：
Falaschi, A
影响因子：
2.9
作者：
COLLINS, JM;GLOCK, MS;CHU, AK
通讯作者：
CHU, AK
DOI：
10.1073/pnas.91.15.7119
发表时间：
1994-07-19
影响因子：
11.1
作者：
GIACCA, M;ZENTILIN, L;FALASCHI, A
通讯作者：
FALASCHI, A
DOI：
10.1073/pnas.90.16.7769
发表时间：
1993-08-15
影响因子：
11.1
作者：
JIN, YJ;BURAKOFF, SJ
通讯作者：
BURAKOFF, SJ