TY - JOUR
T1 - Origins of Sequence Selectivity in Homologous Genetic Recombination
T2 - Insights from Rapid Kinetic Probing of RecA-mediated DNA Strand Exchange
AU - Lee, Andrew M.
AU - Xiao, Jie
AU - Singleton, Scott F.
N1 - Funding Information:
This work was supported by US National Institutes of Health grant GM58114-07 to S.F.S. The authors dedicate this paper to Professor Peter B. Dervan, valued mentor and advocate, on the occasion of his sixtieth birthday.
PY - 2006/7/7
Y1 - 2006/7/7
N2 - Despite intense effort over the past 30 years, the molecular determinants of sequence selectivity in RecA-mediated homologous recombination have remained elusive. Here, we describe when and how sequence homology is recognized between DNA strands during recombination in the context of a kinetic model for RecA-mediated DNA strand exchange. We characterized the transient intermediates of the reaction using pre-steady-state kinetic analysis of strand exchange using oligonucleotide substrates containing a single fluorescent G analog. We observed that the reaction system was sensitive to heterology between the DNA substrates; however, such a "heterology effect" was not manifest when functional groups were added to or removed from the edges of the base-pairs facing the minor groove of the substrate duplex. Hence, RecA-mediated recombination must occur without the involvement of a triple helix, even as a transient intermediate in the process. The fastest detectable reaction phase was accelerated when the structure or stability of the substrate duplex was perturbed by internal mismatches or the replacement of G·C by I·C base-pairs. These findings indicate that the sequence specificity in recombination is achieved by Watson-Crick pairing in the context of base-pair dynamics inherent to the extended DNA structure bound by RecA during strand exchange.
AB - Despite intense effort over the past 30 years, the molecular determinants of sequence selectivity in RecA-mediated homologous recombination have remained elusive. Here, we describe when and how sequence homology is recognized between DNA strands during recombination in the context of a kinetic model for RecA-mediated DNA strand exchange. We characterized the transient intermediates of the reaction using pre-steady-state kinetic analysis of strand exchange using oligonucleotide substrates containing a single fluorescent G analog. We observed that the reaction system was sensitive to heterology between the DNA substrates; however, such a "heterology effect" was not manifest when functional groups were added to or removed from the edges of the base-pairs facing the minor groove of the substrate duplex. Hence, RecA-mediated recombination must occur without the involvement of a triple helix, even as a transient intermediate in the process. The fastest detectable reaction phase was accelerated when the structure or stability of the substrate duplex was perturbed by internal mismatches or the replacement of G·C by I·C base-pairs. These findings indicate that the sequence specificity in recombination is achieved by Watson-Crick pairing in the context of base-pair dynamics inherent to the extended DNA structure bound by RecA during strand exchange.
KW - base flipping
KW - homologous recombination
KW - pre-steady-state kinetics
KW - stopped-flow fluorescence
KW - transient intermediates
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U2 - 10.1016/j.jmb.2006.04.065
DO - 10.1016/j.jmb.2006.04.065
M3 - Article
C2 - 16756994
AN - SCOPUS:33745279076
SN - 0022-2836
VL - 360
SP - 343
EP - 359
JO - Journal of molecular biology
JF - Journal of molecular biology
IS - 2
ER -