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Journal of Bacteriology, October 2001, p. 5772-5777, Vol. 183, No. 19
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.19.5772-5777.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Genetic Recombination in Bacillus
subtilis 168: Effect of 
helD on DNA Repair
and Homologous Recombination
Begoña
Carrasco,1
Silvia
Fernández,1
Marie-Agnes
Petit,2 and
Juan C.
Alonso1,*
Department of Microbial Biotechnology, Centro
Nacional de Biotecnología, CSIC, Campus Universidad
Autónoma de Madrid, Cantoblanco, 28049 Madrid,
Spain,1 and INRA-Génétique
Microbienne, 78352 Jouy-en-Josas Cedex, France2
Received 18 April 2001/Accepted 9 July 2001
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ABSTRACT |
The B. subtilis 
helD allele
rendered cells proficient in transformational recombination and
moderately sensitive to methyl methanesulfonate when present in an
otherwise Rec+ strain. The helD allele
was introduced into rec-deficient strains representative
of the 
(recF strain), 
(addA addB), 
(recH), 
(recU), and 
(recS) epistatic groups. The helD
mutation increased the sensitivity to DNA-damaging agents of
addAB, recU, and
recS cells, did not affect the survival of
recH cells, and decreased the sensitivity of
recF cells. helD also partially suppressed the DNA repair phenotype of other mutations classified within the epistatic group, namely the recL,
recO, and recR mutations. The
helD allele marginally reduced plasmid transformation (three- to sevenfold) of mutations classified within the , , and
epistatic groups. Altogether, these data indicate that the loss of
helicase IV might stabilize recombination repair intermediates formed
in the absence of recFLOR and render
recFLOR, addAB, and recH cells impaired in plasmid transformation.
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TEXT |
Genetic analysis using
chromosomal and plasmid transformation allows the classification of
Bacillus subtilis Rec
strains, other
than recA strains, within five different epistatic groups
(, , , , and groups) (6, 12). Unless
otherwise stated, the indicated genes and products are of B. subtilis origin. The epistatic group activity requires the
recF, recL, recO, recR, and
recN genes (3-5). The recF,
recL, recO, and recR strains and the
Escherichia coli recF
(recFEco),
recOEco, and
recREco strains have similar phenotypes
and share indirect suppressors (e.g., a special type of recA
mutant) (2, 10). It was assumed therefore that RecFLOR has
its counterpart in the RecFOREco complex
(6, 13). The epistatic group activity is dependent on
the addA and addB genes (3). The
addA and addB genes encode different subunits of
the nuclease-helicase AddAB (also termed exonuclease V or
RecBCDEco for E. coli)
(8, 9, 16). The epistatic group activity requires the
recP and recH genes (6). The epistatic group activity requires the recB, recD,
and recU genes (4), and the group is
dependent on recS, a gene sharing homology with
recQ of both B. subtilis and E. coli origin (12). The function whose mutants were
classified within the , , and epistatic groups does not seem
to have a counterpart in E. coli (6, 14).
Identification of the helD gene.
DNA helicases
are involved in both the generation of the recombinogenic
substrates and branch migration of synapsed Holliday junctions
(16). RecBCDEco and the
recQEco,
uvrDEco, and
helDEco gene products have been implicated
in the RecBCDEco and
RecFEco recombination pathways,
respectively. The RecBCD DNA helicase is implicated in the presynaptic
stage of recombination. Furthermore, it has been suggested that
the presence of RecQEco,
UvrDEco, or E. coli
helicase IV (helD gene product, a 3' to 5' DNA helicase) is
required for efficient recombination and repair in a
recBCEco
sbcB(C)Eco
background (21, 23). The B. subtilis pcrA,
addAB, and yvgS genes encode a DNA helicase or
have a high degree of identity with bona fide DNA helicases of the SF1
family (8, 9, 16, 24, 25), and the recS and
recQ gene products have identity with DNA helicases of the
SF2 family, according to the nomenclature of Gorbalenya and Koonin
(15). No UvrDEco,
RepEco, or
HelEco counterparts have been described
for B. subtilis (17, 24).
Helicases of the SF1 family share seven conserved motives scattered
along the protein sequence. Structural analysis has shown that these
motives come close together in the folded protein, bind ATP, and
correspond to the translocating domain of these helicases, allowing the
tracking of single-stranded DNA through the protein. The seven motives
delimit four domains in the primary sequence of the SF1 helicases, an
N-terminal domain preceding the first motive, a 1B domain between the
second and third motives, a 2B domain between the fifth and sixth
motives, and a C-terminal (C-t) domain after the last motive (Fig.
1). An analysis of the length of these
four domains in any helicase of the SF1 family allows their
classification into one of the three following groups: (i) the PcrA
subfamily, helicases with a fixed 1B and 2B length (~134 and 220 to
240 residues, respectively); (ii) the
HelDEco subfamily, helicases with a long
C-t and a short 2B domain; and (iii) the AddA subfamily, helicases with
a long C-t domain. The four helicases of the SF1 family in B. subtilis and in E. coli were classified accordingly, as
shown Fig. 1. PcrA and YjcD belonged to the first group, together with
UvrDEco and
RepEco. The YvgS protein belonged to the
second group, together with HelDEco or
E. coli helicase IV. We therefore propose to name the YvgS protein HelD. It should be noted, however, that the overall level of
similarity between HelD and HelDEco is low
(22% identity). AddA and RecBEco belonged
to the third group. Again, these last two proteins have only 21.7%
identity, but they have been shown to be functionally equivalent
(8, 9).
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FIG. 1.
Classification of DNA helicases of the SF1 family from
B. subtilis and E. coli into three
subfamilies. The seven conserved motives are shown with boxes separated
by the four domains, called N-t (N-terminal), 1B, 2B, and C-t
(C-terminal). The distances (in amino acid residues) separating the
different clusters of motives are reported on the right part of the
figure. See the text for details.
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PcrA, which is able to suppress the UV sensitivity defect of a
uvrDEco mutant, is an essential DNA
helicase (
24). A defect
in RecS decreases plasmid
transformation ~100-fold when present
in an otherwise
Rec
+ strain (
12). The AddAB protein
has its counterpart in the RecBCD
Eco enzyme (
8,
9,
16). Nothing is known about the role of
RecQ
and helicase IV (
helD gene product) in recombination and
DNA
repair. Here, we report the genetic analysis of a
helD null
allele (
helD) in combination with
rec
function, classified within
the
,
,
,
, and
epistatic
groups. We show that the loss
of helicase IV stabilized recombination
repair intermediates formed
in the absence of
recFLOR and
rendered
recFLOR,
addAB, and
recH cells impaired in plasmid transformation. Furthermore, the fact
that
the
helD mutation is a common partial suppressor of the
recFLOR mutations further supported the classification of
the
recFLOR genes within the
epistatic
group.
Disruption of the helD gene by use of a selectable
marker.
It has been shown previously that (i) E. coli
helicase IV is a DNA helicase (26), (ii) it shares several
biochemical properties with the E. coli helicase II and Rep
proteins (27), (iii) double helicase II
(uvrDEco)-helicase IV
(helDEco) deletion mutants are defective
in DNA recombination and repair (20), (iv) B. subtilis possesses two genes homologous to the
uvrDEco-repEco
tandem, the pcrA and yjcD genes
(reference 24; also see above), (v) PcrA, which suppresses
the UV sensitivity defect of a uvrDEco
mutant, is an essential helicase of B. subtilis, whereas a
yjcD mutant does not exhibit a UV-sensitive phenotype
(24). To learn whether the helD gene is
involved in recombinational DNA repair, we have constructed a
helD deletion strain. The helD gene was
disrupted using the plasmid pMUTIN2 (details of the mutant
construction are available on the Micado site
[http://locus.jouy.inra.fr/cgi-bin/genmic/madbase/progs/madbase.operl]). As a result, the helD gene is disrupted starting 139 bp
downstream from the start codon. The helD mutant allele
was transferred into the B. subtilis chromosome of the wild
type (YB886) and its isogenic rec-deficient derivatives of
groups (recF15 [BG129], recL16 [BG107],
recO [BG439], and recR13
[BG127]) and (addA5 addB72 [BG189]), (recH342 [BG119]), (recU
[BG427]), and (recS [BG425]) listed in Table
1, by a double crossing-over event as
previously described (5). The presence of the desired replacement was confirmed by PCR amplification and nucleotide sequence
analysis (data not shown).
Interaction between helD and the functions
classified within the , , , , and epistatic
groups.
To understand the role of the helD gene product
in homologous recombination, we investigated the genetic interaction
between helD and the following mutant genes:
recF, addA addB (addAB), recH, recU, and recS, which were
selected as representatives of the five different epistatic groups
(, , , , and , respectively). B. subtilis
(rec+) and its isogenic rec-deficient
derivative strains (listed in Table 1) were exposed to the killing
action of DNA-damaging agents as previously described (1).
The removal of lesions from Sn2-type simple alkylating agents, such as
methyl methanesulfonate (MMS), could be carried out to a different
degree by adaptive response and recombination repair (1).
As revealed in Fig. 2, various degrees of
increased sensitivity to 10 mM MMS were observed. The helD and recS cells are slightly sensitive,
the addAB and recH cells are moderately
sensitive, and the recF and recU cells are very sensitive to the killing action of MMS compared to the
rec+ control (1, 5, 12, 13)
(Fig. 2).
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FIG. 2.
Survival of B. subtilis strains following
exposure to 10 mM MMS. Survival of rec+
(filled circles) and helD1 strains (empty circles) is
shown. (A) Survival of recF15 cells (empty squares) and
helD1 recF15 cells (filled squares). (B)
Survival of addA5 addB72 cells (empty
squares) and helD1 addA5
addB72 cells (filled squares). (C) Survival of
recH342 cells (empty squares) and helD1
recH342 cells (filled squares). (D) Survival of
recU1 cells (empty squares) and helD1
recU1 cells (filled squares). (E) Survival of
recS1 cells (empty squares) and helD1
recS1 cells (filled squares). The chemical treatment of
DNA repair-deficient mutant strains was performed essentially as
previously described (1).
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The
helD mutant allele increases the MMS sensitivity of
addAB,
recU, and
recS cells but
does not affect the survival rate
of
recH cells (Fig.
2B to
E). Previously we have shown that the
recS null allele
partially suppressed the sensitivity of
addAB cells to MMS
(
12). Since
helD slightly increases the
sensitivity
of
addAB and
recS cells, we have
to assume that helicase IV operates
at a different stage than the AddAB
and RecS DNA helicases. Furthermore,
if a helicase is needed at the
presynaptic stage in the RecF pathway,
as suggested by some experiments
with
E. coli (
21), the fact
that
helD
addAB and
helD recS cells are more
proficient in
recombinational DNA repair than
recF and/or
recU cells (Fig.
2) suggests that helicase IV is not the
only helicase responsible
for presenting the DNA substrate to RecA. An
alternative DNA helicase
(e.g., PcrA, YjcD, RecQ, RecG or
RuvAB), alone or in concerted
action with a nuclease, could
generate the recombinogenic substrate.
Because the
pcrA gene
product is essential (
24), the double
mutant
pcrA
helD could not be tested. Whether any of the remaining
putative
helicases, encoded by
yjcD,
recG,
recQ, or
ruvAB, is
involved at this stage remains
to be
determined.
The
helDEco cells do not exhibit a
UV-sensitive phenotype (
21). In
B. subtilis the
removal of purine adducts produced by
4-nitroquinoline-1-oxide
(4NQO) has been reported to involve nucleotide
excision repair
and recombination repair (
1). To analyze and
interpret the
involvement of the
helD allele in recombinational
repair,
we have exposed the
helD recF strain to the killing
action
of 100 µM 4NQO.
helD cells are slightly sensitive
to 4NQO (Fig.
3), but when the
helD recF strain was exposed to the killing
action of
4NQO a suppression of the
recF defect was observed (Fig.
3).
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FIG. 3.
Survival of B. subtilis strains following
exposure to 100 µM 4NQO. Survival of rec+
cells (filled circles), helD1 cells (empty circles),
recF15 cells (empty squares), and helD1
recF15 cells (filled squares) is shown.
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From these data we can infer that (i)
helD renders cells
slightly sensitive to DNA-damaging agents, (ii) the
helD
gene product
(helicase IV) contributed to different extents to the
removal
of DNA damage in
addAB (group
),
recH
(group
),
recU (group
), and
recS
(group
) cells, and (iii) the absence of helicase
IV partially
suppressed the recombinational repair deficiency
of
recF cells.
helD partially suppresses the DNA repair defects
of the recF, recL, recO,
and recR mutants.
Previously we have shown that
suppressors of the recF defect (e.g., recA73
mutant, overexpression of ssb product) also suppressed the
recL, recO, and recR defects (2,
13). To address whether the helD allele also
suppressed the defect of the recL, recO, and
recR mutations, the respective double mutant strains have been constructed (Table 1).
Unlike the case with
recBCEco
sbcB(C)
Eco cells, which show a
synergistic interaction between the
helDEco gene and
the
recFEco or
recOEco gene for the repair of MMS-damaged
DNA
(
23), the
helD allele suppressed the
recombinational repair
defect of
recF,
recL,
recR, and
recO cells (Fig.
2A,
3, and
4).
Similar results were observed when
the mutant cells were challenged
with 100 µM 4NQO (Fig.
3 and data
not shown).
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FIG. 4.
Survival of B. subtilis strains following
exposure to 10 mM MMS. Survival of rec+
(filled circles) and helD1 (empty circles) strains is
analyzed. (A) Survival of recL16 cells (empty squares)
and helD1 recL16 cells (filled squares).
(B) Survival of recO1 cells (empty squares) and
helD1 recO1 cells (filled squares). (C)
Survival of recR13 cells (empty squares) and
helD1 recR13 cells (filled squares).
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The introduction of a plasmid-borne
helD gene in the
helD recF background resulted in a phenotype
indistinguishable from
that of
recF cells (data not
shown). It is likely, therefore,
that
helD is a bona fide
suppressor of the
recFLOR mutants and
that the absence of
helicase IV redirects recombinational repair
into an avenue different
from the RecFLOR
complex.
Effect of helD on genetic recombination.
To
study the effect of the helD mutation on homologous DNA
recombination, we analyzed the requirement of the helD
function in natural transformational recombination. Natural
transformation in B. subtilis involves the transfer of naked
double-stranded DNA from the media, with the subsequent degradation of
one of the exogenous DNA strands by a set of competence
(com) genes (11, 18, 19). Under these
conditions, the level of expression of the recA and
addAB genes is also increased. The recipient competent uninucleated cell takes up the other DNA strand in a linear
single-stranded form (see references 11, 18, and
19 for reviews). Therefore, by the activity of the
com genes, the donor single-stranded DNA is presented to the
recombinational machinery in a form that it is ready for a homology
search on the parental molecule. Hence, in our analysis we are studying
the late presynaptic, synaptic, and postsynaptic stages but neglect the
involvement of rec functions in the presentation of the
substrate (early presynaptic stage) (14).
Except in
recA cells where chromosomal transformation is
blocked (~10
4-fold reduction), chromosomal
transformation did not change more
than fourfold relative to the
wild-type value when present in
an otherwise wild-type strain (1, Table
2). The effect of double
or triple
mutations in some cases, however, is drastic. A
recFLOR (epistatic group
) mutation blocks (>1,000-fold reduction)
chromosomal
transformation of
addAB (group
) and
recH (group
) cells and
reduces (>20-fold) that of
recU (group
) or
recS (group
)
cells
(
2,
6,
12,
13). A
recH mutation blocks
(>1,000-fold)
chromosomal transformation of
addAB cells and
reduces (>200-fold)
that of
recS cells, but it does not
affect chromosomal transformation
of
recU cells more than
fourfold (
5,
6,
12,
13).
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TABLE 2.
Effect of the helD mutation on homologous
recombination as measured by transformation of chromosomal and
plasmid DNAa
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Plasmid establishment on transformation of
B. subtilis
competent cells is dependent on the degree of oligomerization of the
plasmid genome and requires the RecU, RecO, and RecS products
(
7,
12,
13) but is independent of the RecA activity (
12,
18,
19). Except in the
recO,
recU, and
recS strains (50-fold
reduction), the frequency of
plasmid transformation did not change
more than twofold relative to the
wild-type value (
12,
13).
A
recFLOR mutation
blocks (>100-fold) plasmid transformation of
addAB (group
) and
recH (group
) cells and reduces (>15-fold)
that
of
recU or
recS cells (
5,
6,
12,
13). A
recH mutation reduced plasmid transformation
of
recU and
recS cells
about 10-fold
(
12). Altogether those data indicate that the
gene
products classified with the
,
,
,
, and
epistatic
groups provide overlapping activities that compensate for the
effects of a single
mutation.
By measuring both chromosomal (intermolecular recombination) and
plasmid (intramolecular recombination) transformation we
can examine
different types of events.
B. subtilis competent cells
were
transformed with 1 µg of homologous chromosomal DNA or plasmid
DNA/ml
to determine the transformation frequency of the
Rec
mutant strains. The frequency of appearance
of
met+ transformants in the single Rec
strains and in certain double
Rec
strains has been previously
reported (
1). Here, however,
the experiments were
performed in parallel for comparison with
other strains (Table
2). We
show here that
helD deletion did
not affect more than
threefold chromosomal transformation of
recF,
addAB,
recH,
recU, and
recS cells (Table
2).
The
helD deletion did not affect more than twofold
plasmid transformation of the highly impaired
recU and
recS cells, and
it reduced three- to sevenfold plasmid
transformation of
recF,
addAB, and
recH cells (Table
2).
What is the role for helicase IV in DNA recombination?
The
AddAB, PcrA, helicase IV, RecS, and RecQ helicases are the orthologs of
the presynaptic RecBCDEco,
UvrDEco, E. coli
helicase IV, and RecQEco DNA
helicases, respectively. However, genetic studies on mutants of these
various helicases start to uncover some differences in terms of their
respective roles in repair and recombination in the two hosts. PcrA is
essential in B. subtilis, whereas
UvrDEco is not essential under laboratory growth conditions (24). It has been shown that
RecQEco is required for conjugational
recombination and DNA repair in a
recBCEco
sbcB(C)Eco background
(22), whereas the RecS helicase partially suppressed the
DNA repair defect of addAB cells and was required for
plasmid transformation and DNA repair in an otherwise Rec+
background (12). The
uvrDEco,
helDEco,
recQEco, and helD
cells were recombination and repair proficient when present in
otherwise Rec+ cells (21, 22, this work). A
helDEco mutation, which has no effect
on repair and recombination in an
recBCEco sbcB(C)Eco background,
interacts synergistically with recFEco
and recOEco in the repair of
MMS-damaged DNA (21), but we show here that
helD partially suppresses the DNA repair defect of
recFLOR cells.
A possible way to reconcile these contrasting observations is to
consider that in
B. subtilis recombinational repair takes
place mostly through the functions classified within the
(
recFLOR)
and
(
recB,
recU, and
recD) epistatic groups. To illustrate
this
point, one should compare the steepness of MMS curves for a
recF or a
recU mutant to the modest effect of MMS
on survival of an
addAB mutant. Furthermore, genetic studies
are made possible in
a RecF
Add
+ background in
B. subtilis which
are prevented in
E. coli given
the preponderance of the
RecBCD pathway, hiding all possible effect
of mutations on the RecF
pathway. Taking these data into account,
we propose that in a
recF background, helicase IV extends the
number of gaps
created upon injury to DNA, which results in a
severe sensitivity to
genotoxic agents. When it is absent, fewer
gaps are created, which are
taken in charge by the functions classified
within the
(RecB, RecU,
and RecD) and
(AddAB) epistatic groups,
and this results in the
partial suppression of the repair defect.
Such an effect cannot be
observed in
E. coli for the above-mentioned
reasons (work in
a
recBC sbcBC background). Still, one observation
made for
E. coli could be reminiscent of such a role for
helicase
IV. It was found that the double
recQEco uvrDEco
mutant could
not be constructed in a
recBCEco sbcBCEco
background, whereas
the triple
recQEco
uvrDEco helDEco mutant
was successfully constructed,
albeit with difficulty (
22).
This suggests that somehow the
absence of helicase IV in this mutant
had a positive effect, possibly
by reducing the amount of
substrate that RecFOR had to deal with.
A similar and
symmetrical argument can be drawn in the case of
recS
(
12). In conclusion, studies on recombination in
B. subtilis are instructive and stimulating for the broadening of our
view
on recombination mechanisms in bacteria at
large.
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ACKNOWLEDGMENTS |
This research was supported by grant BMC2000-0548 from MCyT-DGI to
J.C.A.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbial Biotechnology, Centro Nacional de Biotecnología,
CSIC, Campus Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain. Phone: (34) 91585 4546. Fax: (34) 91585 4506. E-mail:
jcalonso{at}cnb.uam.es.
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Journal of Bacteriology, October 2001, p. 5772-5777, Vol. 183, No. 19
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.19.5772-5777.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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