Zinc affects the conformation of nucleoprotein filaments formed by replication protein A (RPA) and long natural DNA molecules.

Replication protein A is the major single strand DNA binding protein of human cells, composed of three subunits with molecular weights of 70, 32, and 14 kDa. Most of the DNA binding activity of RPA has been mapped to the largest subunit that contains two OB-fold DNA binding domains and a third, OB-like structure in the carboxyterminal domain (CTD). This third domain resembles an OB-fold with a zinc binding domain inserted in the middle of the structure, and has recently been shown to carry a coordinated Zn(II) ion. The bound metal ion is essential for the tertiary structure of the RPA70-CTD, and appears to modulate its DNA binding activity when tested with synthetic oligonucleotides. We show here that zinc strongly affects the conformation of nucleoprotein filaments formed between RPA and long natural DNA molecules. In these experiments, the CTD is dispensable for DNA binding and the unwinding of long double stranded DNA molecules. However, using band shift assays and electron microscopy, we found that RPA-DNA complexes contract at zinc concentrations that do not affect the conformations of complexes formed between DNA and a RPA70 deletion construct lacking the CTD. Our data suggest that nucleoprotein complexes with RPA in its natural, zinc-bearing form may have a compact rather than an extended conformation.

DNA binding properties of a 110 kDa nucleolar protein.

A single strand specific DNA binding protein was purified to homogeneity from calf thymus nucleoprotein. The monomeric protein is elongated in shape and has a molecular mass of 110 kDa. Since immunocytochemistry revealed that the protein is predominantly located in the nucleolus we refer to it as the 110 kDa nucleolar protein. The protein binds not only to single stranded DNA but also to single stranded RNA, including homopolymeric synthetic RNA. We have used the single stranded DNA binding properties of the 110 kDa protein in model studies to investigate its effects on the configuration of nucleic acid. Our results are: only 50-55 protein molecules are sufficient to saturate all binding sites on the 6408 nucleotides of phage fd DNA; protein binding cause a compaction of single stranded DNA; large nucleoprotein aggregates are formed in the presence of divalent cations; this is due to protein-protein interactions which occur at moderately high concentrations of magnesium-, calcium or manganese ions; the protein induces the reassociation of complementary nucleic acid sequences. We speculate that the 110 kDa protein performs similar reactions in vivo and may have a function related to the processing and packaging of preribosomal RNA.

Reactions in vitro of the DNA polymerase-primase from Xenopus laevis eggs. A role for ATP in chain elongation.

A form of DNA polymerase alpha was purified several thousandfold from a protein extract of Xenopus laevis eggs. The enzyme effectively converts, in the presence of ribonucleoside triphosphates, a circular single-stranded phage fd DNA template into a double-stranded DNA form and, therefore, must be associated with a DNA primase. We first show by gel electrophoresis in the presence of sodium dodecyl sulfate that both enzymatic activities, DNA polymerase and primase, most probably reside on a greater than 100 000-Da subunit of the DNA polymerase holoenzyme. We then assayed the polymerase-primase at various template/enzyme ratios and found that the DNA complementary strand sections synthesized in vitro belong to defined size classes in the range of 600-2000 nucleotides, suggesting preferred start and/or stop sites on the fd DNA template strand. We show that the stop sites coincide with stable hairpin structures in fd DNA. We have used a fd DNA template, primed by a restriction fragment of known size, to show that the polymerase-primase stops at the first stable hairpin structure upstream from the 3'-OH primer site when the reaction was carried out at 0.1 mM ATP. However, at 2 mM ATP the enzyme was able to travers this and other stop sites on the fd DNA template strand leading to the synthesis of 2-4 times longer DNA strands. Our results suggest a role for ATP in the polymerase-primase-catalyzed chain-elongation reaction.