How the cell can mend broken DNA using another DNA copy as template has
puzzled researchers for years. How is it possible to find the correct
sequences in the busy interior of the cell? Researchers from Uppsala
university have now discovered the solution; it is easier to find a rope
than a ball if you are blindfolded.
When a DNA molecule breaks in two, the fate of the cell is threatened. From
the perspective of a bacterium, fixing the break quickly is a matter of life
and death. But to mend the DNA without introducing mistakes in the sequence
is challenging; the repair machinery needs to find a template. The process
of healing broken DNA using a template from a sister chromosome is known as
homologous recombination and is well described in the literature.
However, the description usually disregards the daunting task of finding the
matching template among all the other genome sequences. The chromosome is a
complex structure with several million base pairs of genetic code and it is
quite clear that simple diffusion in 3D would not be sufficiently fast by a
long shot. But then, how is it done? This has been the mystery of homologous
recombination for 50 years. From previous studies, it is clear that the
molecule RecA is involved and important in the search process, but, up until
now, this has been the limit of our understanding of this process.
Using a CRISPR-based technique
Now, a group of Uppsala researchers headed by Professor Johan Elf has
finally found the solution to this search enigma. In a study that is
published in Nature, they use a CRISPR-based technique to make controlled
DNA breaks in bacteria. By growing the cells in a microfluidic culture chip
and tracking labeled RecA molecules with fluorescence microscopy, the
researchers can image the homologous recombination process from start to
finish.
“The microfluidic culture chip allows us to follow the fate of thousands of
individual bacteria simultaneously and to control CRISPR-induced DNA breaks
in time. It is very precise, almost like having a pair of tiny DNA
scissors,” says Jakub Wiktor, one of the researchers behind the study.
The label on RecA together with fluorescent markers on the DNA allows the
researchers to follow every step of the process accurately; for example,
they conclude that the whole repair is finished in 15 minutes, on average,
and that the template is located in about nine.
Rearranging RecA to form thin filaments
Using microscopy, Elf and his team investigate the fate of the break site
and its homologous copy in real-time. They also find that the cell responds
by rearranging RecA to form thin filaments that span the length of the cell.
“We can see the formation of a thin, flexible structure that protrudes from
the break site just after the DNA damage. Since the DNA ends are
incorporated into this fiber, it is sufficient that any part of the filament
finds the precious template and thus the search is theoretically reduced
from three to two dimensions. Our model suggests that this is the key to
fast and successful homology repair,” says Arvid Gynnå, who has worked on
the project throughout his PhD studies.
Might help us understand the causes of tumor growth
Going from a 3D to a 2D search is indeed a considerable improvement
regarding the probability of finding the homologous sequence quickly enough,
or in fact, at all. As the Japanese mathematician, Shizuo Kakutani put it:
“A drunk man will find his way home, but a drunk bird may be lost forever”.
With these words, he tried to explain a curious fact; an object that
explores a 2D surface by a random walk will sooner or later find its way
back to its starting point while in a 3D space, it is likely that it will
never return “home”.
The Uppsala researchers performed their study in the model organism E. coli,
but the process of homology repair is nearly identical for higher organisms
such as ourselves, or doves for that matter. DNA damage occurs frequently in
our bodies, and without the ability to heal broken DNA, we would be
extremely vulnerable to, for example, UV light and reactive oxygen species,
and more likely to develop cancer. In fact, most oncogenes are related to
DNA repair and the new mechanistic insights might help us understand the
causes of tumor growth.
Reference:
Wiktor, J., Gynnå, A.H., Leroy, P. et al. RecA finds homologous DNA by reduced
dimensionality search. Nature (2021).
DOI: 10.1038/s41586-021-03877-6