The first step in the likely mechanism for the generation of the Holliday junction (Figure 6.37) involves
formation of a single stranded nick in one of the DNA duplexes. This leads to strand invasion and forma-
tion of a D loop with the displaced strand. The enzymes in E. colithat can catalyse this single strand nick-
ing and strand invasion process are known.58,59Nicking is achieved by the RecBCDenzyme complex, a
large protein complex of about 3 105 Da mass. RecBCD can only bind to a free DNA duplex end, but
once bound, it moves along the duplex, unwinding the helix as it goes and rewinding the DNA behind it.
If RecBC encounters a specific sequence termed a Chi siteas it moves along the DNA, it cuts 56 bases 3
to it. RecBC continues to unwind the DNA but the rewinding is prevented by the nick. This leaves a
single-stranded region that participates in strand invasion. This process is catalysed by the RecA protein.^60
RecA binds to the single-stranded region and inserts it into a DNA duplex with which it is homologous. In
this way, a combination of RecA and RecBCD proteins can catalyse the formation of a Holliday junction.
240 Chapter 6
Figure 6.37 Involvement of the RecBCD and RecA proteins in the generation of the Holliday junction
Figure 6.36 The Holliday junction is an intermediate in homologous recombination