Related Experiment Video

Updated: Aug 7, 2026

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis
11:08

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

Published on: June 19, 2018

Mutations in conserved yeast DNA primase domains impair DNA replication in vivo

S Francesconi1, M P Longhese, A Piseri

  • 1Dipartimento di Genetica e di Biologia dei Microrganismi, Milano, Italy.

Proceedings of the National Academy of Sciences of the United States of America
|May 1, 1991
PubMed

Insights

This study demonstrates the essential role of eukaryotic DNA primase in DNA replication. Conditional mutations in yeast DNA primase genes confirmed its necessity for synthesizing high molecular weight DNA products in vivo.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Eukaryotic DNA primase is crucial for initiating DNA replication.
  • Understanding its in vivo function is essential for comprehending replication fidelity and mechanisms.

Purpose of the Study:

  • To investigate the in vivo role of eukaryotic DNA primase.
  • To generate and characterize conditional and lethal mutations in yeast DNA primase subunits.

Main Methods:

  • Random in vitro mutagenesis of PR11 and PR12 genes encoding yeast DNA primase subunits.
  • Replacement of wild-type genes with conditional alleles (pri1 and pri2).
  • Analysis of DNA synthesis and product formation at restrictive temperatures.

Main Results:

  • Conditional yeast strains exhibited altered DNA synthesis upon temperature shift.
  • Reduced ability to synthesize high molecular weight DNA products was observed.
  • Nucleotide-level mapping revealed conserved amino acid residue changes in mutated subunits.

Conclusions:

  • This study provides in vivo evidence for the essential function of DNA primase in eukaryotic DNA replication.
  • Mutations affecting conserved amino acid residues highlight critical functional domains of DNA primase.

More Related Videos

Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis09:04

Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis

Published on: July 26, 2018

Strand-Specific Analysis of Proteins at Replicating DNA Strands by Enrichment and Sequencing of Protein-Associated Nascent DNA Method08:53

Strand-Specific Analysis of Proteins at Replicating DNA Strands by Enrichment and Sequencing of Protein-Associated Nascent DNA Method

Published on: May 2, 2025

Related Concept Videos

DNA Replication02:40

DNA Replication

DNA replication involves the separation of the two strands of the double helix, with each strand serving as a template from which the new complementary strand is copied.  After replication, each double-stranded DNA includes one parental or “old” strand and one “new” strand. This is known as semiconservative replication. The resulting DNA molecules have the same sequence and are divided equally into the two daughter cells.
Replication in Prokaryotes
DNA replication uses a large number of...
The DNA Replication Fork01:02

The DNA Replication Fork

An organism’s genome needs to be duplicated in an efficient and error-free manner for its growth and survival. The replication fork is a Y-shaped active region where two strands of DNA are separated and replicated continuously. The coupling of DNA unzipping and complementary strand synthesis is a characteristic feature of a replication fork.   Organisms with small circular DNA, such as E. coli, often have a single origin of replication; therefore, they have only two replication forks, one in...
Translesion DNA Polymerases02:10

Translesion DNA Polymerases

Translesion (TLS) polymerases rescue stalled DNA polymerases at sites of damaged bases by replacing the replicative polymerase and installing a nucleotide across the damaged site. Doing so, TLS allows additional time for the cell to repair the damage before resuming regular DNA replication.
TLS polymerases are found in all three domains of life - archaea, bacteria, and eukaryotes. Of the different classes of TLS polymerases, members of the Y family are fitted with specialized structures that...
Homologous Recombination02:31

Homologous Recombination

The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
Restarting Stalled Replication Forks02:37

Restarting Stalled Replication Forks

DNA replication is initiated at sites containing predefined DNA sequences known as origins of replication. DNA is unwound at these sites by the minichromosome maintenance (MCM) helicase and other factors such as Cdc45 and the associated GINS complex.The unwound single strands are protected by replication protein A (RPA) until DNA polymerase starts synthesizing DNA at the 5’ end of the strand in the same direction as the replication fork. To prevent the replication fork from falling apart, a...
Conservative Site-specific Recombination and Phase Variation02:53

Conservative Site-specific Recombination and Phase Variation

Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...