Image provided by Sue Amundsen.
Genetic recombination is the swapping of DNA between two different sources, for example between similar sequences of DNA from two different organisms or between sequences of DNA within the genome of one organism. It is a widely used mechanism to repair DNA damaged by environmental mutagens or by errors that occur during replication. Recombination happens near some DNA sequences more frequently than others and these special sites are termed "hotspots". Specific hotspots were discovered in certain mutants of the bacterial virus, lambda phage, that require recombination for reproduction. Phages with these hotspots, called Chi for crossover hotspot instigator (abbreviated χ), grow better in their host, the bacterium Escherichia coli (E. coli). 35 years ago scientists in Dr. Smith’s Lab, now in the Fred Hutch Basic Sciences Division, identified the Chi hotspot sequence as 5'-GCTGGTGG-3'.
E. coli employs a multi-protein enzyme complex called RecBCD to carry out recombination by unwinding and cutting one DNA strand (called "nicking") at Chi. The structure of RecBCD bound to dsDNA revealed that a "tunnel" in RecC appears to directly recognize the Chi sequence. In recent publications in Genetics and Nucleic Acids Research, scientists in the Smith Lab challenge the previous conclusion that Chi is limited to 5'-GCTGGTGG-3' and offer new insight into how the enzyme drives homologous recombination (HR).
The scientists began by creating recC "tunnel mutants" designed to disrupt the recognition of the Chi sequence but not compromise the overall structure or activity of the RecBCD complex. They measured the ability of recC mutant E. coli to recognize Chi by comparing the growth of recombination-deficient lambda phage without a Chi site (χo) or with a Chi site (χ+76) at one site in their genome. They characterized many recC mutant E. coli strains, and found around 25 that could not support growth of lambda phage with or without a Chi site. These recC mutants were still able to carry out recombination instigated through the high-frequency recombination (Hfr) pathway that occurs when bacteria mate with each other and exchange pieces of DNA. Thus, the mutants appeared to be specifically unable to recognize Chi.
In order to identify whether the mutants could now identify a new Chi-like sequence, defined by the amino acids in their RecC tunnel, the scientists created a "library" of recombination-deficient lambda phage, each strain with 25 nucleotides (nt) of random sequence inserted into a new location in their genome. As expected based on calculations, they observed that approximately one out of 3000 phage strains were able to form large plaques (colonies) when grown with E. coli. All of the phage that formed large plaques on wild-type, recBCD+ E. coli contained the Chi sequence at the insertion locus, as expected. When the scientists sequenced the insert of the phage that formed large plaques on RecC tunnel mutant E. coli, they expected to identify Chi-like sequences that were recognized by the altered RecC tunnel. To their surprise, however, they found that all had the Chi sequence. This suggested that a Chi sequence at a different locus in the phage genome could be recognized by these recC mutants while the originally tested χ+76 locus was not recognized. Indeed, they observed good phage growth in the RecC mutants when the phage had a Chi site inserted at several other loci in their genome. This shows that the RecC tunnel mutants are able to recognize the Chi sequence but that Chi hotspot activity depends on the genomic context of Chi.
To assess how the surrounding sequence affected Chi hotspot activity, the scientists inserted the Chi site with four randomized nucleotides on either side into a new locus. By swapping the sequence to the right (3') or left (5') of the Chi sequence, they found that the sequence to the right, but not to the left, of the Chi sequence strongly dictates hotspot (recombination) activity.
Purified RecBCD nicks dsDNA at Chi, so the scientists designed a method to identify which surrounding sequence instilled "hotter" hotspot activity. They created a library of short lengths of DNA (oligonucleotides, called oligos for short) containing the Chi sequence each flanked by a random sequence and incubated each oligo in the library with a low concentration of RecBCD. They purified oligos cut by RecBCD and deep sequenced them. By comparing which sequences were cut versus uncut, they were able to determine that the sequence to the left (5') of Chi plays little role in influencing RecBCD activity. Intriguingly, they found a strong preference for RecBCD to cut oligos with purines (A or G) 4-7 nucleotides to the right (3') of Chi. Remarkably, the RecB nuclease cuts at these same positions. Their results demonstrate that the activity of recombination-promoting enzymes such as RecBCD requires more complex interactions with the DNA than had been appreciated.
Decades after identifying the Chi sequence, Dr. Smith smiles at the serendipity of research. "If you keep your eyes open , you’ll always find something new."
Amundsen SK, Sharp JW, Smith GR. 2016. "RecBCD enzyme "Chi recognition" mutants recognize Chi recombination hotspots in the right DNA context." Genetics. In press.
Taylor AF, Amundsen SK, Smith GR. 2016. "Unexpected DNA context-dependence identifies a new determinant of Chi recombination hotspots." Nucleic Acids Research. In press.
This research was funded by the National Institutes of Health.