Direct observations of DNA are giving new insights into how
genetic material is copied and repaired.
"We can monitor the process directly, and that gives us a
different perspective," said Roberto Galletto, a postdoctoral
scholar at UC Davis and first author on a paper published Sept. 20
on the Web site of the journal Nature.
In E. coli bacteria, molecules of an enzyme called RecA attach
themselves along a DNA strand, stretching it out and forming a
filament. A piece of complementary DNA lines up along side it, and
pieces of DNA can be swapped in to repair gaps in the original
strand. A similar protein, called Rad51, does the same job in
humans.
"How RecA and Rad51 assemble into filaments determines the
outcome of DNA repair, but very little is known about how assembly
is controlled," said senior author Stephen Kowalczykowski, professor
in the sections of Microbiology and of Molecular and Cellular
Biology and director of the Center for Genetics and Development at
UC Davis. Genes that control the human gene, Rad51, have been linked
to increased risk of breast cancer.
Galletto attached a short piece of DNA to a tiny latex bead and
placed it in a flow chamber, held by laser beam "tweezers." Fluid
flowing past made the DNA stream out like a banner. Then he nudged
it into an adjacent channel containing fluorescently-tagged RecA.
After short intervals of time, he moved it back to the first chamber
to observe the results.
By repeatedly dipping the same piece of DNA into the fluorescent
channel, the researchers could see the RecA form clusters of four to
five molecules on the DNA. Once those clusters had formed, the
DNA/RecA filament rapidly grew in both directions. The measurements
made in those experiments will be the baseline for future studies of
both RecA and Rad51, Kowalczykowski said.
The new work adapts an approach developed by Kowalczykowski and
Ronald J. Baskin, professor of molecular and cellular biology, to
study single enzymes at work unwinding DNA strands. That research
was first published in Nature in 2001.
In addition to Galletto, Kowalczykowski and Baskin, the research
team included postdoctoral scholar Ichiro Amitani. The work was
funded by the National Institutes of Health and a fellowship awarded
to Galletto by the Jeane B. Kempner Foundation.