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Directionality of Replication Fork Movement Determined by Two-Dimensional Native–Native DNA Agarose Gel Electrophoresis

  • Andreas S. Ivessa
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1054)

Abstract

The analysis of replication intermediates by the neutral–neutral two-dimensional agarose gel technique allows determining the chromosomal positions where DNA replication initiates, whether replication forks pause or stall at specific sites, or whether two DNA molecules undergo DNA recombination events. This technique does not, however, immediately tell in which direction replication forks migrate through the DNA region under investigation. Here, we describe the procedure to determine the direction of replication fork progression by carrying out a restriction enzyme digest of DNA imbedded in agarose after the completion of the first dimension of a 2D gel.

Key words

Replication intermediates Chromosomal DNA DNA replication Direction of replication fork movement In-gelo Yeast 

References

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    Friedman KL, Brewer BJ (1995) Analysis of replication intermediates by two-dimensional agarose gel electrophoresis. Methods Enzymol 262:613–627PubMedCrossRefGoogle Scholar
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    Brewer BJ, Lockshon D, Fangman WL (1992) The arrest of replication forks in the rDNA of yeast occurs independently of transcription. Cell 71:267–276PubMedCrossRefGoogle Scholar
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    Ivessa AS, Lenzmeier BA, Bessler JB, Goudsouzian LK, Schnakenberg SL, Zakian VA (2003) The Saccharomyces cerevisiae helicase Rrm3p facilitates replication past nonhistone protein–DNA complexes. Mol Cell 12:1525–1536PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2013

Authors and Affiliations

  • Andreas S. Ivessa
    • 1
  1. 1.Department of Cell Biology and Molecular MedicineUniversity of Medicine and Dentistry of New JerseyNewarkUSA

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