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Mechanisms of Mutation Group

Mutagenesis in E. coli

Roel M. Schaaper, Ph.D.
Roel Schaaper, Ph.D.
Principal Investigator
Tel (919) 541-4250
Fax (919) 541-7613
schaaper@niehs.nih.gov
P.O. Box 12233
Mail Drop E3-01
Research Triangle Park, NC 27709

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Research Summary

Roel M. Schaaper, Ph.D., heads the Mechanisms of Mutation Group within the Genome Integrity and Structural Biology Laboratory.

Using the E. coli model system, the Mechanisms of Mutation Group studies the mechanisms of mutagenesis. The following four studies are ongoing.

  1. Chromosomal DNA replication fidelity
    This project investigates the fidelity of the Pol III holoenzyme replication complex (HE) that is responsible for simultaneously replicating the leading and lagging strands at the replication fork. In particular, this study is focused on the roles of the 17 individual subunits of HE in determining the fidelity of the HE complex and the mechanisms underlying the observed differences in the fidelity of leading-strand and lagging-strand replication.
  2. Role of the deoxynucleoside triphosphate (dNTP) pools in determining mutation rates
    The dNTPs are important factors in determining polymerase error rates, and this project aims to better understand the rules underlying their effects. In particular, the investigations focus on (i) the mutational consequences of dNTP disturbances, (ii) the isolation of novel mutator and antimutator mutants affected in dNTP metabolism, and (iii) analysis of potential sanitizing activities that may remove modified dNTP derivatives from the pools in vivo.
  3. Mechanisms of base analog mutagenesis
    A novel detoxification pathway has been discovered for certain N-hydroxylated base analogs such as N6-hydroxylaminopurine (HAP) and N4-hydroxycytosine. These agents are strongly mutagenic in the absence of the detoxification pathway. It has been established that this pathway requires the molybdenum cofactor, but the precise mechanism of detoxification is unclear. Therefore, this project is focused on investigating the genetic and biochemical basis of this novel process.
  4. Structural analysis of Pol III core subunits
    Pol III core is composed of three subunits: α (the 5'-->3' polymerase), ε (the 3'-->5' proofreader) and θ, the small (8 kD) subunit. These are tightly bound in the order, α- ε-θ. The function of θ is largely unknown. To better understand its function and its interaction with the ε subunit, structural studies are being performed using multidimensional NMR methods. As part of this project studies are also carried out on the bacteriophage P1 hot gene product, a homolog of E. coli θ that can substitute for θ.
Novel mutators of E. coli carrying a defect in the dnaX gene.  As shown here, these mutator colonies of E. coli contain a large number of blue spots (papillae), giving the colony an overall blue appearance, while normal non-mutator colonies display only a few such spots.  Each papilla represents a lac+ subclone within the starting (white) lac- colony, and the rate with which such subclones are produced during the growth of the colony is an indicator of the mutation rate in a growing colony.  The mutator colonies contain a mutation in the dnaX gene, encoding the Tau subunit of DNA polymerase III which replicates the bacterial chromosome.  The discovery of these mutants reveals the important role of the Tau subunit in determining the fidelity of DNA replication.
Novel mutators of E. coli carrying a defect in the dnaX gene.

 

Major areas of research:

  • Mechanisms of mutagenesis
  • Fidelity of DNA replication
  • Sanitation of cellular deoxynucleoside triphosphate pools

Current projects:

  • Investigation of the DNA Polymerase III holoenzyme complex (HE), focusing on the role of its subunits and the mechanisms underlying observed differential fidelity of leading and lagging strands
  • Investigation of the role of the deoxynucleoside triphosphate (dNTP) pools in determining mutation rates
  • Characterization of the genetic and biochemical features of a novel detoxification pathway for N-hydroxylated base analogs
  • Structural analysis of Pol III core subunits

Schaaper received his Ph.D. in mathematics and natural sciences from the University of Leiden, The Netherlands, in 1983. He has published 80 peer-reviewed articles in leading biomedical journals, as well as several book chapters.

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