Proteins
of Recombination
The Holliday and Meselson-Radding model of recombination (here) can easily be adapted to explain daughter strand gap repair or the recombination that occurs in bacteria after transformation or conjugation. Some of the proteins thought to participate in bacterial recombination include the following:
1. DNA polymerase;
2. DNA ligase;
3. Single-strand DNA-binding protein;
4. RecA protein--a multifunctional protein with Mr of about 38,000. In recombination it promotes the pairing of homologous strands (see here). Several strand-pairing reactions can be demonstrated in vitro. Two examples are shown in Figure 25.23. RecA binds just 3 DNA strands during the strand exchange reaction that it catalyzes. A model for the strand exchange reaction is shown in Figure 25.24. In the process, RecA wraps single-stranded DNA (Figure 25.23, Figure 25.24) and then examines double-stranded DNA for sequences complementary to those in the single strand by sliding the single stranded DNA along the double stranded DNA in a process that requires ATP. When complementarity is found, branch migration occurs, with simultaneous strand exchange;
5. Exonuclease V (also called RecBCD nuclease)--In E. coli, recombination occurs preferentially at or near the octanucleotide sequence, 5'-GCTGGTCC-3'. This sequence is called Chi. The RecBCD protein (a heterotrimer of the protein products of the recB, recC, and recD genes) displays sequence specificity for Chi. It binds at a double-strand break on duplex DNA and uses a helicase activity to unwind and partially degrade the DNA. The 3' end is displaced as a loop, which is coated with single strand binding protein. When the enzyme reaches Chi, a sequence-specific interaction causes RecBCD to switch strands and switch its preferred polarity of DNA degradation. These changes facilitate the loading of RecA to a free 3' end, which initiates strand invasion of a nearby duplex (Figure 25.28);
6. ruvA--a DNA binding protein, whose specificity directs it toward the four-stranded Holliday structure;
7. ruvB--an ATP-requiring "motor protein," which binds to two opposed arms of the Holliday junction and rotates them in opposite directions, forcing branch migration by driving the rotational movement of the other two strands toward the junction; and
8. ruvC--begins resolution of the Holliday structure by nicking two strands.
Ruv protein homology has not yet been detected in eukaryotic cells, but much of the mechanism appears to be similar to that of E. coli. For example, the RAD51 protein of human cells and yeast has a strand-pairing activity similar to that of RecA.
In eukaryotic cells, an essential function of homologous recombination is the repair of double-strand breaks. A broken eukaryotic chromosome can use the sequence information in its homolog to reconstruct the original DNA sequence at the site of the break (Figure 25.30).
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