Muprints and Whole Genome Insertion Scans: Methods for Investigating Chromosome Accessibility and DNA Dynamics using Bacteriophage Mu.

Bacteriophage Mu infects a broad range of gram-negative bacteria. After infection, Mu amplifies its DNA through a coupled transposition/replication cycle that inserts copies of Mu throughout all domains of the folded chromosome. Mu has the most relaxed target specificity of the known transposons (Manna et al., J Bacteriol 187: 3586-3588, 2005) and the Mu DNA packaging process, called "headful packaging", incorporates 50-150 bp of host sequences covalently bound to its left end and 2 kb of host DNA linked to its right end into a viral capsid. The combination of broad insertion coverage and easy phage purification makes Mu ideal for analyzing chromosome dynamics and DNA structure inside living cells. "Mu printing" (Wang and Higgins, Mol Microbiol 12: 665-677, 1994; Manna et al., J Bacteriol 183: 3328-3335, 2001) uses the polymerase chain reaction (PCR) to generate a quantitative fine structure map of Mu insertion sites within specific regions of a bacterial chromosome or plasmid. A complementary technique uses microarray platforms to provide quantitative insertion patterns covering a whole bacterial genome (Manna et al., J Bacteriol 187: 3586-3588, 2005; Manna et al., Proc Natl Acad Sci U S A 101: 9780-9785, 2004). These two methods provide a powerful complementary system to investigate chromosome structure inside living cells.

Measuring In Vivo Supercoil Dynamics and Transcription Elongation Rates in Bacterial Chromosomes.

DNA gyrase is the only topoisomerase that can catalytically introduce negative supercoils into covalently closed DNA. The enzyme plays a critical role in many phases of DNA biochemistry. There are only a few methods that allow one to measure supercoiling in chromosomal DNA and analyze the role of gyrase in transcription and its interaction with the other three bacterial topoisomerases. Here, we provide molecular tools for measuring supercoil density in the chromosome and for connecting the dots between transcription and DNA topology.