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Homologous pairing in vitro stimulated by the recombination hotspot, Chi.

Authors
Type
Published Article
Journal
Cell
Publisher
Elsevier
Volume
66
Issue
2
Pages
361–371
Source
Kowalczykowski Lab
License
Unknown

Abstract

Genetic recombination in Escherichia coli is stimulated at DNA sequences known as Chi sites, 5 -GCT-GGTGG-3 . We describe the in vitro formation of homologously paired joint molecules that is dependent upon this recombination hotspot. Chi-dependent joint molecule formation requires RecA, RecBCD, and SSB proteins and a Chi site in the donor linear dsDNA. The donor dsDNA is unwound by RecBCD enzyme, and the invasive strand is generated by nicking at Chi. This Chi-dependent invading strand must contain homology to the recipient supercoiled DNA substrate at its newly formed 3 end for efficient joint molecule formation. Action at Chi generates invasive ssDNA from the 5 but not the 3 side of Chi, suggesting that the nuclease activity of RecBCD enzyme is attenuated upon encountering a Chi site. These results support the view that RecBCD enzyme action can precede RecA protein action and reconcile the seemingly opposing degradative and recombination functions of RecBCD enzyme.

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