Abstract
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.
Highlights
Circular single-stranded phage M13 DNA is used as a template for complementary strand synthesis in cytosolic extracts from proliferating HeLa cells
We found that radioactively labeled desoxynucleotides were efficiently incorporated into reaction products thatmigrate in agarose gels as dsDNA (Fig. 1).Most of the the synthesis products appearedbetorelaxed or slightly supercoiled circular double-stranded products, but a fraction had thelectrophoretic properties of linear dsDNA (Fig. 1).About 120pg of unfractionated cytosolic proteins was sufficient to convert 60 pmol(nucleotides) of the ssDNA template intoradioactively labeled double-stranded products, DNA synthesis was not observedwhen M13 dsDNA was added as a template to cytosolic extracts
We found that the Complementary strand DNA synthesis differs from semi- formation of highly supercoiled DNA was promoted by aliconservative SV40 DNA replication in a number of aspects, quots of fractions 3-6 (Fig. 6D), with fraction5 inducing the and it ibsy no means certain that the mechanismofsnucleo- most highly supercoiled reaction products
Summary
From the Fakultatfur Biologie, Uniuersitat Konstanz, 0-7750 Konstanz, Federal Republic of Germany. Regular nucleosome spacing on dsDNA can be achieved when the in vitro assembly reaction is performed in unfractionated cell extracts from Xenopus eggs and oocytes (Laskey and Earnshaw, 1980; Glikin et al, 1984) This requires the activity of a protein kinase modifying a histone H2A variant as one possible condition for regular spacing (Kleinschmidt and Steinbeisser, 1991). In high speed supernatants of Xenopus egg extracts, complementary DNA strandsare synthesized on added single-stranded DNA, concomitantly with nucleosome assembly (M6chali and Harland, 1982) This process appears to proceed ina two-step reaction like that with nonreplicating dsDNA (Dilworth et al, 1987),but replicationassociated chromatin assembly seems to be kinetically favored (Almouzni and Mkchali, 1988; Almouzniet al., 1990). Sumably newly synthesized histones, present in the cytosolic extract, on replicating SV40 DNA (Smithand Stillman, 1991a) and SV40 minichromosomes (Krude et al, 1993) in vitro
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