Single-strand DNA intermediates in phage λ’s Red recombination pathway

Single-strand DNA intermediates in phage λ’s Red recombination pathway

^*Institute of Molecular Biology, University of Oregon, Eugene, OR 97403-1229; and ^†Laboratory of Microbial Structure and Function, National Institutes of Health, National Institute of Allergy and Infectious Disease, Rocky Mountain Laboratories, Hamilton, MT 59840-2999

Abstract

An assay was developed to assess early intermediates arising in λ’s Red recombination pathway. Double-strand breaks were delivered in vivo to nonreplicating λ chromosomes. Analysis by blot hybridization of total DNA extracts revealed the following: (i) long (>1.4 kilobases) single-strand DNA (ssDNA) intermediates; (ii) resection proceeding bidirectionally from the break site; (iii) single-strand overhangs of 3′ polarity; and (iv) in the absence of λ’s ninR functions, a requirement of the redα gene product for the production of ssDNA. Therefore, the physical characteristics exhibited by these ssDNA molecules are consistent with their being an early recombination intermediate in the Red recombination pathway as proposed previously from genetic and in vitro biochemical analyses.

Footnotes

↵ ‡ Deceased January 22, 1996.
↵ § To whom reprint requests should be addressed. e-mail: fstahl{at}molbio.uoregon.edu.
Franklin W. Stahl
ABBREVIATIONS:

DSB,

double-strand break;

ssDNA,

single-strand DNA