Insight into the Human DNA Primase Interaction with Template-Primer

DNA replication in almost all organisms depends on the activity of DNA primase, a DNA-dependent RNA polymerase that synthesizes short RNA primers of defined size for DNA polymerases. Eukaryotic and archaeal primases are heterodimers consisting of small catalytic and large accessory subunits, both of...

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Bibliographic Details
Published in:The Journal of biological chemistry 2016-02, Vol.291 (9), p.4793-4802
Main Authors: Baranovskiy, Andrey G., Zhang, Yinbo, Suwa, Yoshiaki, Gu, Jianyou, Babayeva, Nigar D., Pavlov, Youri I., Tahirov, Tahir H.
Format: Article
Language:English
Subjects:
5′-triphosphate
Binding Sites
DNA and Chromosomes
DNA primase
DNA Primase - chemistry
DNA Primase - genetics
DNA Primase - metabolism
DNA replication
DNA, Single-Stranded - chemistry
DNA, Single-Stranded - metabolism
DNA-Directed DNA Polymerase - chemistry
DNA-Directed DNA Polymerase - genetics
DNA-Directed DNA Polymerase - metabolism
DNA-protein interaction
Electrophoretic Mobility Shift Assay
Fluorescence Polarization
Fluorescent Dyes - chemistry
gel electrophoresis
human
Humans
Kinetics
Models, Molecular
Multifunctional Enzymes - chemistry
Multifunctional Enzymes - genetics
Multifunctional Enzymes - metabolism
Nucleic Acid Conformation
p58 subunit
Peptide Fragments - chemistry
Peptide Fragments - genetics
Peptide Fragments - metabolism
primase activity
Protein Conformation
Protein Interaction Domains and Motifs
Protein Multimerization
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
RNA - chemistry
RNA - metabolism
RNA primer length counting
RNA synthesis
RNA/DNA duplex
Transcription, Genetic
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Summary:DNA replication in almost all organisms depends on the activity of DNA primase, a DNA-dependent RNA polymerase that synthesizes short RNA primers of defined size for DNA polymerases. Eukaryotic and archaeal primases are heterodimers consisting of small catalytic and large accessory subunits, both of which are necessary for the activity. The mode of interaction of primase subunits with substrates during the various steps of primer synthesis that results in the counting of primer length is not clear. Here we show that the C-terminal domain of the large subunit (p58C) plays a major role in template-primer binding and also defines the elements of the DNA template and the RNA primer that interact with p58C. The specific mode of interaction with a template-primer involving the terminal 5′-triphosphate of RNA and the 3′-overhang of DNA results in a stable complex between p58C and the DNA/RNA duplex. Our results explain how p58C participates in RNA synthesis and primer length counting and also indicate that the binding site for initiating NTP is located on p58C. These findings provide notable insight into the mechanism of primase function and are applicable for DNA primases from other species.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M115.704064