Nucleosome assembly during complementary DNA strand synthesis in extracts from mammalian cells.
Circular single-stranded phage M13 DNA is used as a template for complementary strand synthesis in cytosolic extracts from proliferating HeLa cells. DNA synthesis is initiated by one or maximally two priming events and typically leads to covalently closed double-stranded reaction products. When carried out in the presence of the nuclear chromatin assembly factor CAF-1, complementary strand synthesis is accompanied by nucleosome assembly. This novel system is very useful for the study of basic biochemical aspects concerning the assembly of nucleosomes. The activity of CAF-1 completely depends on complementary strand synthesis and acts stoichiometrically to promote the assembly of nucleosomes in a noncooperative manner. Apparently, CAF-1 activity is coupled to DNA synthesis via a structural feature of replicating DNA, most likely its partial single strandedness.
Krude
T
T
Fakultät für Biologie, Universität Konstanz, Federal Republic of Germany.
Knippers
R
R
D017104:Bacteriophage M13 / Q000235:genetics / Q000378:metabolism; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000378:metabolism / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis* / Q000302:isolation & purification / Q000648:ultrastructure; D004261:DNA Replication; D004270:DNA, Circular / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008854:Microscopy, Electron; D009707:Nucleosomes / Q000378:metabolism* / Q000648:ultrastructure; D013698:Templates, Genetic
D004270:DNA, Circular; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D009707:Nucleosomes; D004247:DNA