JOURNAL OF BACrERIOLOGY, May 1970, p. 404-410
Copyright a 1970 American Society for Microbiology
Dark-Recovery Processes in Escherichia coli
Irradiated with Ultraviolet Light
III. Effect of rec Mutations on Recovery of Excision-Deficient
Mutants of Escherichia coli K-12
ANN K. GANESAN AND KENDRIC C. SMITH
Department ofRadiology, Stanford University School of Medicine, Stanford, California 94305
Received for publication 29 December 1969
Mutants of Escherichia coli K-12 unable to excise pyrimidine dimers from their
deoxyribonucleic acid (DNA) because of a uvr mutation show a higher survival
when plated on a minimal salts medium after exposure to ultraviolet radiation
than when plated on a complex medium such as nutrient agar containing yeast ex-
tract. This response has been called minimal medium recovery (MMR). Recovery
of uvr mutants can take place in liquid as well as on solid medium, but not in buffer
or under conditions of amino acid starvation that do not permit cell growth and
normal DNA replication. MMR can thus be distinguished from the recovery of
recombination-deficient (rec- uvr+) derivatives of K-12 which can occur under
conditions where growth is not possible. Because MMR is characteristic of excision-
defective mutants, it evidently reflects a type of repair independent of excision.
We have obtained genetic evidence that MMR is determined by the rec genes,
which also control recombination in K-12. Cells carrying a uvr mutation together
with recA13, recA56, recB21, or recC22 failed to show MMR and were more sensi-
tive to ultraviolet radiation than either their rec+ uvr- or rec- uvr+ parents. The
rec+ uvr- derivatives obtained from recA uvr- strains by transduction or by rever-
sion regained the capacity for MMR. Our results indicate that inactivation of any
one of the three genes, recA, recB, or recC, prevents cells from showing MMR.
One means by which Escherichia coli can re-
cover from the effects of ultraviolet (UV) radia-
tion is to excise pyrimidine dimers produced in
its deoxyribonucleic acid (DNA) by irradiation,
replacing them with normal DNA synthesized by
using the undamaged complementary region of
the opposite strand as template (see 9, 11, 20, 21
for reviews). The uvrA, uvrB, and uvrC mutants
of E. coli K-12 are deficient in their capacity for
excision. They are thus unable to repair DNA
by this mechanism and are more sensitive to UV
radiation than corresponding uvr+ strains (1, 13).
However, such mutants appear to be able to re-
cover from UV damage by some other process.
When irradiated and plated on minimal medium,
they show a higher survival than when plated on
a complex medium, such as nutrient agar con-
taining yeast extract. This response, called mini-
mal medium recovery (MMR), indicates that the
cells can recover on minimal medium and that
their recovery is inhibited by complex medium
(6, 7).
The recombination-deficient (rec) mutants of
K-12 examined, except recC22, showed a similar
response (6). In contrast to the uvr (rec+) mu-
tants, however, the rec (uvr+) mutants could also
recover in buffer or in minimal medium lacking
amino acids needed for cell growth. The difference
in the conditions required suggested that recovery
of the uvr mutants reflected a different mechanism
of repair than the one underlying the recovery of
rec mutants (7). In addition, genetic evidence
indicated that the recovery of rec mutants was
inactivated by uvr mutations (8). Thus, the re-
covery shown by the uvr mutants must involve a
system different from the one responsible for the
recovery of rec mutants.
The rec uvr recombinants which had been
tested showed little or no recovery on minimal
medium, indicating that the rec genes might con-
trol the recovery of excision-deficient cells on
minimal medium (7). However, we felt that more
data relating to this possibility was needed. With
the exception of one strain, all of the rec uvr
derivatives examined had been constructed from
the same Hfr recA parent. Matings had been
404
Vol. 102, No. 2
Printed In U.S.A.

RECOVERY OF EXCISION-DEFICIENT MUTANTS
made with several different thy F- strains. In
each case, Thy+ recombinants were selected (8).
Thus, it was possible that the lack of recovery
in the recA uvr isolates from these crosses might
have resulted from a mutation in a gene linked to
thy but different than recA. In addition, no re-
combinants carrying a recB or recC mutation
together with a uvr marker had been tested, so
that the effect of these genes on recovery was un-
known. The experiments described in this paper
were designed to provide more information re-
garding the role of the rec genes in the recovery
of excision-deficient mutants.
MATERIALS AND METHODS
Most of the procedures have been described previ-
ously (6-8).
Strains. The derivatives of E. coli used are described
in Table 1.
Media. Complex medium included YENB (0.75%
Difco yeast extract, 2.3% Difco nutrient agar) and
YENB liquid (0.75% Difco yeast extract, 0.8%
Difco nutrient broth).
The phosphate-buffered minimal media used have
been described previously (6).
L-Amino acids were incorporated to a final con-
centration of 10-3 M: thymine, 10 ,ug/ml; thiamine,
0.5 jug/ml; and dihydrostreptomycin sulfate, 200
pg/ml.
Irradiation. An unfiltered 25-w General Electric
Germicidal lamp was used at a distance of 54 cm from
the surface of a platform shaker. Two perforated
grills were used to adjust the dose rate to 820 ergs
per mm2 per min (for doses above 100 ergs/mm2)
and 26 ergs per mm2 per min (for doses below 100
ergs/mm2) as measured by the photodecomposition
of potassium ferrioxalate (10, 17).
Reversion. Selection based on UV resistance was
used to obtain Rec+ revertants. Single-colony isolates
were grown overnight in Penassay broth (Difco Anti-
biotic Medium 3). Samples of 0.1 ml were spread on
YENB agar, incubated for 30 min, and then irradiated
with 12 ergs/mm2. After 2 days of incubation, colonies
were picked and tested for UV sensitivity, recombina-
tion, and host-cell reactivation, as previously de-
scribed (6).
RESULTS
Preliminary experiments. Experiments were
undertaken to determine what conditions were
suitable for measuring recovery in excision-
deficient (uvr) derivatives of K-12, and to com-
pare the responses of uvr and recombination-
deficient (rec) mutants under these conditions.
The uvr (rec+) mutants, in contrast to rec
TABLE 1. Escherichia coli K-12 derivatives useda
Designa- Mating
tion type
W3110
AB1884
AB1885
AB1886
AB2480
AB2487
AB2498
AB2499
AB2500
JC1569
JC2918
JC2926
JC5088
JC5489
JC5495
JC5743
SR58
SR80
SR87
F-
F-
F-
F-
?-
F-
F-
F-
F-
F-
F-
F-
F-
F-
F-
F-
F7-
Relevant
genotype
avrC34
uvrB5
uvrA6
recAJ3 uvrA6
recA13
uvrC34
uvrBS
uvrA6
recAl
recA56
recAS6
recC22
recAJ3 recB21
recB21
recAS6 uvrBS
recC22 uvr BS
recB21 uvrBS
Other markers
Xta
X' thr leu arg his pro ara lac gal mtl xyl strr T6r thi
XI' thr leu arg his pro ara lac gal mtl xyl strr T6r thi
XI' thr leu arg his pro ara lac gal mtl xyl strr 6r thi
X' pro thi lac gal strr T6r
XI thr leu arg his pro thy ara lac gal mtl xyl strr T6r thi
XI thr leu arg his pro thy ara lac gal mtl xyl strr T6r thi
X' thr leu arg his pro thy ara lac gal mtl xyl strr T6r thi
X'I thr leu arg his pro thy ara lac gal mtl xyl strr T6r thi
X' thr leu arg his pro ara lac gal mtl xyl strr T6r thi
X' thr leu arg his pro ara lac gal mtl xyl strr T6r thi
X' thr leu arg his pro ara lac gal mtl xyl strr T6r thi
Xs thr ilv thi spMr
Xs thr leu arg his pro ara lac gal mtl xyl strr T6r thi
X' thr leu arg his pro ara lac gal mtl xyl strr T6r thi
X' thr leu arg his pro ara lac gal mtl xyl strr T6r thi
X' thr leu arg his pro (ara lac gal mtl xyl) strr T6r thi
X' thr leu arg his pro (ara lac gal mtl xyl) strr T6r thi
X' thr leu arg his pro (ara lac gal mtl xyl) strr T6r thi
Reference
13
13
13
16
15
13
13
13
2
2
24
a Abbreviations (3, 4, 24): The symbols arg, his, ilv, leu, pro, try, thi, thr, thy denote requirements for
arginine, histidine, isoleucine and valine, leucine, proline, tryptophan, thiamine, threonine, and thy-
mine, respectively; ara, gal, lac, mtl, and xyl denote the inability to utilize arabinose, galactose, lactose,
mannitol, and xylose, respectively; T6, X, spm, and str denote response to the phages T6 and X, and to
the antibiotics, spectinomycin and streptomycin (r indicates resistance, a, sensitivity); rec denotes genes
affecting genetic recombination and UV sensitivity; uvr designates genes affecting host-cell reactivation
and UV sensitivity. Markers in parentheses have not been tested, but are inferred from the characteristics
of the parent strains.
405VOL. 102, 1970