Bacteria fix DNA like mammals
Double-strand DNA break repair pathways appear conserved, suggesting new
ways to make libraries
By Charles Q Choi

Bacteria and mammals may use similar mechanisms in a major yet poorly
understood DNA repair pathway, suggests a report in the October 22 Science.

In theory, coauthor Aidan Doherty of the University of Sussex in Brighton
told The Scientist, this system-uses of which are the subject of a patent
filed by the University of Cambridge-should enable the cloning of any DNA
fragments, regardless of the structure of the ends, and has great potential
for the generation of random DNA libraries.

Non-homologous end joining (NHEJ) is the main pathway for resection and
repair of DNA double-strand breaks (DSBs) with incompatible ends in
mammalian cells. The mechanisms required in NHEJ are poorly understood. Many
DSBs require processing by polymerases and nucleases to produce ligatable
termini. The Mycobacterium tuberculosis DNA repair ligase, Mt-Lig, has
domains exhibiting significant homology with polymerases and possibly
nucleases, suggesting it might prove a model for NHEJ.

Doherty's team found that purified recombinant Mt-Lig was an efficient
DNA-dependent DNA polymerase in template-dependent primer extension assays
and similarly acted as a DNA-dependent RNA polymerase. The researchers also
found Mt-Lig possessed 3' to 5' single-strand DNA exonuclease activity,
progressively digesting the 3' but not the 5' single strand tails of partial
duplexes until reaching the double-strand region.

In addition, in polymerization assays with oligonucleotides that produce a
nonligatable 1-nt gap upon alignment, Mt-Lig efficiently filled in the gap
with no detectable strand displacement synthesis. Mt-Lig used adenosine
triphosphate (ATP) and, to a lesser extent, dATP to extend single-strand
DNA, demonstrating terminal transferase activity, which is implicated in
NHEJ.

The researchers also found that Mt-Lig could join aligned DNA duplexes
possessing a 1-nt 3' flap adjacent to a 3-nt gap. Sequencing of ligated
junctions in equivalent assays with substrates with a 3-nt flap adjacent to
a 5-nt gap showed the microhomology sequence was retained and bases
complementary to the template strand replaced the mismatched flap.

"It's just amazing how there are so many activities in one protein. It
should prove quite interesting to dissect it to find out how its component
parts can catalyze so many different events typically separated in
eukaryotes," Steve Kowalczykowski of the University of California at Davis,
who did not participate in this study, told The Scientist.

Full Text at TheScientist
http://www.biomedcentral.com/news/20041025/01

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