1022-7954/00/3612- $25.00 © 2000
Russian Journal of Genetics, Vol. 36, No. 12, 2000, pp. 1370–1374. Translated from Genetika, Vol. 36, No. 12, 2000, pp. 1629–1633.
Original Russian Text Copyright © 2000 by Glasunov, Glaser.
Linearized plasmids with a double-strand gap
(DSG), a deletion at the site of a double-strand break
(DSB), are repaired efﬁciently in
cells via recombination with chromosomal DNA
[1–3]. In Rad
cells, DSG repair is accompanied by the
complete restoration of the previously eliminated
sequence of plasmid DNA.
Earlier, we showed that the plasmid DNA repair in
S. cerevisiae rad57-1
mutant occurs without the res-
toration of the sequence lost because of the DSG [3–5].
This repair mechanism, which we designated as a
homology-dependent ligation, requires the presence of
plasmid DNA cohesive ends, just as in the case of sim-
ple ligation; however, this mechanism depends on the
homology between terminal sites of the disrupted plas-
mid and chromosomal DNA. Thus, a mutation in the
gene leads to triggering a minor mechanism of
DSB repair, which does not lead to the restoration of
plasmid DNA DSG. The reasons for this phenomenon
are still unknown. As for the
gene, its product
is known to be a component of the recombinosomic
complex involved in recombination and recombina-
tional DNA repair .
The Rad57 protein forms a heterodimer with the
gene product. This heterodimer promotes DNA
strand exchange by Rad51 recombinase . In this con-
nection, this work was devoted to clarifying the role of
gene in plasmid DNA DSG repair and com-
paring the results with the corresponding data on the
gene. We found that the cold-sensitive
mutation markedly decreased the precision of DSG
repair under restrictive conditions (23
C): DSG was
repaired in only 5–7% of plasmids, whereas under per-
missive conditions (36
C), DSG was repaired in
approximately 50% of the cells. In the cold-sensitive
mutant, the proportion of plasmids in which
DSGs were repaired was nearly equal under both per-
missive and restrictive conditions and ranged from 5 to
10%. The results indicate that when DSG repair occurs
under restrictive conditions, mutations in both
genes lead to a major contribution of the
mechanism of homology-dependent ligation to the
repair of double-strand DNA breaks.
MATERIALS AND METHODS
We used the following
a leu2 his3 lys2-25
a leu2 his3
a leu2 rad57-1 his3 lys2-25
a leu2 rad57-1 his3 lys2-29
rad55-3 his3 lys2-25.
Strains 2B-D241 and 2G-D252 were provided by
D.A. Gordenin (St. Petersburg State University).
Strains DC5725, DC5729, and DC5525 were con-
structed in the course of this study.
An autonomously replicating bifunc-
tional plasmid pLL12 carries two yeast markers,
, and the
sequence of plasmid pBR322
. Figure 1 shows the plasmid map. The
contains a unique
I restriction site and one
site. The second
HI site is located in the pBR322
Repair of Double-Strand DNA Gaps
Homology-Dependent Ligation and the Role of the
A. V. Glasunov
and V. M. Glaser
State Research Institute of Genetics and Selection of Industrial Microorganisms, Moscow, 113545 Russia;
fax: (095) 315-05-01; e-mail: firstname.lastname@example.org
Department of Genetics and Selection, Moscow State University, Moscow, 119899 Russia
Received June 6, 2000
—In our previous works, a mutation in the
gene was shown to induce the plasmid DNA dou-
ble-strand gap (DSG) repair via a special recombinational repair mechanism: homology-dependent ligation
responsible for reuniting disrupted plasmid ends without reconstructing the sequence lost because of the DSG.
In this work, the role of the
gene in the plasmid DNA DSG repair was studied. A cold-sensitive
mutation markedly decreased the precision of plasmid DNA DSG repair under conditions of restrictive temper-
C): only 5–7% of plasmids can repair DSG, whereas under permissive conditions (36
C), DSGs were
repaired in approximately 50% of the cells. In the cold-sensitive mutation
, the proportion of plasmids
in which DSGs were repaired was nearly the same under both permissive and restrictive conditions (5–10%).
The results indicate that a disturbance in the function of the
gene, as in the
gene, leads to a dras-
tic increase in the contribution of homology-dependent ligation to the repair of double-strand DNA breaks.