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Journal of Bacteriology, July 2007, p. 5101-5107, Vol. 189, No. 14
0021-9193/07/$08.00+0     doi:10.1128/JB.00409-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Effect of a recD Mutation on DNA Damage Resistance and Transformation in Deinococcus radiodurans{triangledown} ,{dagger}

Matthew D. Servinsky and Douglas A. Julin*

Department of Chemistry and Biochemistry, and Program in Molecular and Cell Biology, University of Maryland, College Park, Maryland

Received 20 March 2007/ Accepted 4 May 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The bacterium Deinococcus radiodurans is resistant to extremely high levels of DNA-damaging agents such as UV light, ionizing radiation, and chemicals such as hydrogen peroxide and mitomycin C. The organism is able to repair large numbers of double-strand breaks caused by ionizing radiation, in spite of the lack of the RecBCD enzyme, which is essential for double-strand DNA break repair in Escherichia coli and many other bacteria. The D. radiodurans genome sequence indicates that the organism lacks recB and recC genes, but there is a gene encoding a protein with significant similarity to the RecD protein of E. coli and other bacteria. We have generated D. radiodurans strains with a disruption or deletion of the recD gene. The recD mutants are more sensitive than wild-type cells to irradiation with gamma rays and UV light and to treatment with hydrogen peroxide, but they are not sensitive to treatment with mitomycin C and methyl methanesulfonate. The recD mutants also show greater efficiency of transformation by exogenous homologous DNA. These results are the first indication that the D. radiodurans RecD protein has a role in DNA damage repair and/or homologous recombination in the organism.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The bacterium Deinococcus radiodurans is one of the most resistant to ionizing radiation (IR) and other DNA-damaging agents of all known organisms (2, 6, 39). D. radiodurans DNA is extensively fragmented after treatment with IR, but the broken chromosomes can be reassembled, and the organism survives except at very high radiation doses (2, 6, 49). D. radiodurans is also resistant to UV light and a variety of chemicals that damage DNA. The ability to survive extensive DNA damage is believed to be a consequence of resistance to desiccation, a condition that also leads to oxidative damage to DNA (35).

Several factors are believed to contribute to the extraordinary DNA damage resistance of D. radiodurans. The organism has multiple genome copies (21, 22), which presumably facilitates DNA repair via homologous recombination (49). In addition, its condensed nucleoid structure may keep broken DNA ends aligned for facile repair (31, 51). D. radiodurans also accumulates Mn2+ to high intracellular concentrations and has a low concentration of internal Fe2+. Manganese ion is thought to act as an antioxidant to prevent oxidative damage to cellular components (7, 8, 17).

Much recent research has been directed at identifying and studying the DNA repair enzymes and pathways in D. radiodurans. Repair of double-strand breaks such as those which arise from IR or during replication of a damaged DNA template generally occurs via homologous recombination in bacteria (26-28, 38). The broken DNA ends are first attacked by a helicase/nuclease enzyme (RecBCD in Escherichia coli and AddAB in Bacillus subtilis) (5). The nuclease and helicase activities of the RecBCD enzyme process the broken DNA ends, and the RecBCD enzyme loads the RecA protein onto the resulting 3'-terminated single-stranded DNA strand. The RecA single-stranded DNA filament then binds to a homologous DNA duplex and initiates DNA strand exchange and recombination, leading ultimately to the repair of the DNA break.

Repair of IR-induced DNA damage in D. radiodurans is RecA dependent, since recA mutants are highly sensitive to IR and other DNA-damaging agents (9, 19). Interestingly, there are no identifiable homologues of genes that would encode either the RecB or RecC proteins in the sequenced D. radiodurans genome, nor are there genes related to addAB (48). However, the organism has a gene annotated as recD, which encodes a protein with amino acid sequence similarity to the RecD protein from E. coli and other bacteria. The function of the D. radiodurans RecD protein is unknown, since RecD proteins in other organisms have no known function except as part of the RecBCD enzyme.

RecD proteins share amino acid sequence motifs that are found in the enzymes grouped as superfamily I helicases (18). Genes that encode proteins closely related to RecD from E. coli have been identified in a number of bacterial genomes and are grouped in a single group of orthologues in the Clusters of Orthologous Groups database (accession number COG0507) (see http://www.ncbi.nlm.nih.gov/COG/) (45). Further analysis of these RecD protein sequences shows that they can be divided into two subfamilies based on the amino acid sequences N terminal to the conserved helicase motifs (42, 47). One subfamily (referred to as RecD1 in reference 42) includes RecD subunits of RecBCD enzymes. The second subfamily (RecD2 in reference 42) includes the D. radiodurans RecD protein and closely related proteins from a number of other bacteria including B. subtilis, Lactococcus lactis, Streptococcus pyogenes, and Chlamydia species (42, 47). Most of these bacteria lack a RecBCD enzyme and instead have the AddAB enzyme. D. radiodurans is unusual in that it has a RecD-like protein but neither RecBCD nor AddAB.

In previous work, we purified the D. radiodurans RecD protein and found that it is a DNA helicase capable of efficient unwinding of short double-stranded DNA (dsDNA) substrates (47). In this work, we have generated recD mutations in the D. radiodurans chromosome by insertional mutagenesis. The recD mutants are sensitive to irradiation with gamma rays and UV light and to treatment with hydrogen peroxide, but they are resistant to mitomycin C (MMC) and to methyl methanesulfonate (MMS). A recD mutant is also more transformable than the wild type in assays involving the uptake of DNA from the medium and incorporation into the chromosome by homologous recombination. These results indicate that RecD has a role in the extraordinary DNA damage resistance of D. radiodurans, and they provide the first indication of a role for any member of this RecD protein family in DNA metabolism.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial strains and culture conditions. Wild-type D. radiodurans strains R1 and BAA-816 were obtained from the ATCC, Manassas, VA, and from Michael Daly, Uniformed Services University of the Health Sciences, respectively. D. radiodurans strain LS18 (streptomycin resistant) was a gift from John Battista, Louisiana State University. E. coli strain DH5{alpha} was used for routine cloning procedures. TGY medium contains 0.5% tryptone, 0.3% yeast extract, and 0.1% dextrose. LB medium contains 1% tryptone, 0.5% yeast extract, and 1% NaCl. Antibiotics were added to D. radiodurans cultures as follows: 8 µg/ml kanamycin, 3 µg/ml chloramphenicol, and 50 µg/ml streptomycin unless stated otherwise. Antibiotics were added to E. coli cultures as follows: 30 µg/ml kanamycin and 100 µg/ml ampicillin.

PCRs. PCR mixtures (50 µl) contained 100 ng genomic DNA template or 0.1 ng plasmid DNA template, 0.4 µM primers, and 200 µM deoxynucleoside triphosphates with Pfu DNA polymerase (Stratagene Corp.). Other conditions were as recommended by the DNA polymerase supplier. Primer sequences are listed in Table S1 in the supplemental material. D. radiodurans chromosomal DNA was isolated essentially as described previously (13).

Southern blotting. Southern blotting was performed using genomic DNA isolated from wild-type D. radiodurans or recD mutants as described previously (13). The DNA was digested with appropriate restriction endonucleases (see figure legends) and subjected to agarose gel electrophoresis. DNA was transferred from the gel to uncharged nitrocellulose membranes (Optitran membrane; Schleicher & Schuell) by capillary transfer and fixed by baking at 120°C for 30 min. Digoxigenin-labeled probes were created with the PCR DIG Probe Synthesis kit (Roche). Hybridization was done at 42°C with shaking for 60 min after blocking with salmon sperm DNA, followed by several washes at 60°C. Detection was done using anti-digoxigenin-AP Fab fragments (Roche) and ECF substrate (catalog no. RPN5785; Amersham). Southern blots were visualized using a Storm PhosphorImager (GE Healthcare).

Creation of D. radiodurans recD mutants. Disruption of the recD gene was accomplished by insertional mutagenesis (16). The kanamycin resistance gene and promoter region (kan gene) were PCR amplified from the plasmid pCR-Blunt (Invitrogen Corp.) using primers that introduced SacII and PshAI sites. The D. radiodurans recD gene in plasmid pDr-RecD.ptz (47) and the kan PCR product were digested with PshAI and SacII and ligated to create plasmid pRecDKanDis. Primers complementary to the ends of the recD sequence (DRecD1 and DRecD2) (Fig. 1A and see Table S1 in the supplemental material) were used to PCR amplify the disrupted recD gene (recD::kan gene) using pRecDKanDis as the template. The PCR product was used to transform D. radiodurans strain R1 to kanamycin resistance as described previously (34) (recD::kan mutant 1).


Figure 1
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  		FIG. 1. recD::kan mutant construction and verification. (A) Map of the recD gene region in the wild-type D. radiodurans chromosome (top) and after disruption by insertion of the kanamycin resistance (kan) gene (bottom). The kan gene replaced a 48-bp PshAI-SacII fragment within the recD gene. The recD gene is transcribed left to right, while kan is transcribed right to left, in this diagram. (B) PCRs were done with primers Prom1 and DRecD2 (lanes 1 and 2) and Prom1 and KanFor (lanes 3 and 4) using genomic DNA isolated from either wild-type (wt) or recD::kan mutant (mut) cells. Primer sequences are given in Table S1 in the supplemental material, and their annealing locations are indicated in A. (C) Southern blots. Genomic DNA isolated from the recD::kan mutant was digested with EagI (lane 1), EcoRI (lane 2), ApaI (lane 3), and NcoI/PstI (lane 4) and displayed on a 0.9% agarose gel. The DNA was transferred onto a nitrocellulose membrane, hybridized to a kan gene probe (left-hand panel), stripped, and rehybridized to an undisrupted recD gene probe (right-hand panel). Molecular weights were determined from the signal due to nonspecific hybridization of the digoxigenin-labeled probe to molecular weight markers run in an adjacent lane (see Fig. S1 in the supplemental material). The expected band sizes are 2.3 and 2.5 kb (lane 1), 4.7 kb (lane 2), 4.4 kb (lane 3), and 6.1 and 2.7 kb (lane 4).

 
We generated several versions of the recD::kan mutation to ensure that the phenotypic effects were due to the recD disruption and no other mutation. First, we transferred kanamycin resistance to fresh wild-type cells by transforming D. radiodurans strain BAA-816 with genomic DNA from recD::kan mutant 1 to make recD::kan mutant 2. We then PCR amplified the recD::kan gene using genomic DNA from recD::kan mutant 2 and again transferred it to fresh BAA-816 wild-type cells (recD::kan mutant 3). The results shown in Fig. 2 were obtained with recD::kan mutant 1, while the results in Fig. 3 to 6 were obtained using recD::kan mutant 3. The structure of each recD::kan disruption mutant was confirmed by PCR and by Southern blotting (see Results).


Figure 2
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  		FIG. 2. Growth of wild-type and recD::kan mutant D. radiodurans strains. Cells were grown overnight (with kanamycin for the recD::kan mutant), diluted 1:200 into fresh TGY medium with no antibiotic, and shaken at 30°C. Samples were removed at the indicated times after dilution, and the OD650 was measured with a Perkin-Elmer Lambda Bio spectrophotometer in a 1-cm-path-length cuvette. Closed circles, wild-type strain R1; inverted open triangles, recD::kan mutant.

 

Figure 3
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  		FIG. 3. Sensitivity of the recD::kan mutant to gamma and UV irradiation and to hydrogen peroxide. Closed circles, wild-type strain BAA-816; open inverted triangles, recD mutant. (A) Gamma irradiation was from a 60Co source at 100 Gy/min on ice. (B) Cells were spread onto TGY plates and irradiated with UV light from a germicidal lamp at 90 J/m2/min. (C) Cells were treated with hydrogen peroxide (0.03%; 8.8 mM) for the indicated times and then spread onto TGY plates. The results for gamma and UV irradiation (A and B) are from two independent experiments (n = 6), while the results for hydrogen peroxide are from a single experiment (n = 3).

 

Figure 6
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  		FIG. 6. Test for complementation of the recD::kan disruption mutation by a plasmid-borne recD gene. Sensitivity to irradiation with UV light was measured as described in the legend of Fig. 3 and Materials and Methods. Strains containing the plasmid were grown in the presence of chloramphenicol (3 µg/ml) but were grown without antibiotic after UV irradiation. Closed circles, wild type; open diamonds, wild-type cells transformed with plasmid pRAD1-recD; open inverted triangles, recD::kan mutant; closed squares, recD::kan transformed with pRAD1-recD. The results are representative data (n = 3) from four independent experiments.

 
The recD gene was also deleted completely and replaced by a chloramphenicol resistance gene (cam gene). The cam gene in plasmid p11530 (29) was PCR amplified using Taq DNA polymerase (Fermentas) and ligated into the T/A cloning vector pCR2.1 (Invitrogen Corp.) to make pCR2.1-cam. A 558-bp D. radiodurans chromosomal DNA fragment upstream of the recD gene was amplified by PCR using primers that introduced HindIII and BamHI recognition sites at the upstream and downstream ends, respectively. A 662-bp D. radiodurans chromosomal DNA fragment downstream of the recD gene was amplified by PCR using primers that introduced XhoI and NsiI recognition sites at the upstream and downstream ends, respectively. These two PCR products were digested with HindIII and BamHI, or with XhoI and NsiI, and ligated successively into the pCR2.1-cam plasmid construct cut with the same pairs of restriction endonucleases. This D. radiodurans DNA fragment in pCR2.1 (2,501 bp), with the cam gene replacing the recD gene, was PCR amplified, and the product was used to transform recD::kan mutant 3 (see above) to chloramphenicol resistance, generating the {Delta}recD::cam mutant. The complete deletion of the previously disrupted recD gene was confirmed by the inability of the chloramphenicol-resistant transformants to grow in the presence of kanamycin and by Southern blotting (see Results). The recD coding region is completely deleted in this mutant, along with 104 bp upstream of the ATG start codon and 79 bp downstream of the TAA stop codon.

DNA damage sensitivity assays. Cells for each assay were grown in TGY broth with appropriate antibiotics to mid-log phase (optical density at 600 nm [OD600] of 0.5, corresponding to 2 x 108 cells/ml). Gamma and UV irradiation and MMC sensitivity experiments were done with recD::kan mutants 2 and 3 (see above), with strain BAA-816 as the corresponding wild-type strain. Sensitivity to MMS and hydrogen peroxide was tested only with recD::kan mutant 3.

Sensitivity to gamma irradiation was determined by using a Neutron Products model 200324 60Co source. Cells were irradiated in 1.0-ml portions at about 60 Gy/min in microcentrifuge tubes on ice. Irradiated and mock-irradiated cells were serially diluted in triplicate in TGY medium and spread onto TGY plates without antibiotics. The plates were incubated at 30°C, and colonies were counted after 2 to 3 days.

For UV sensitivity experiments, cells were serially diluted in triplicate in TGY medium and spread onto TGY plates. After the medium was absorbed, the plates were opened and exposed to UV-B light from a 15-W germicidal lamp (FG15T8; Fisher) at a rate of 90 J/m2/min (measured with a UVX Radiometer; UVP, Upland, CA). The plates were incubated at 30°C, and colonies were counted after 2 to 3 days.

Sensitivity to MMC, MMS, or H2O2 was measured by incubating cells in liquid TGY medium containing either 10 µg/ml MMC, 30 mM MMS, or 0.03% H2O2 or with no addition, with shaking at 30°C. At selected times, aliquots from the culture were serially diluted in TGY medium and spread onto TGY plates. The plates were incubated at 30°C, and colonies were counted after 2 to 3 days.

Transformation experiments. Transformation experiments were done according to two different protocols. First, D. radiodurans strains were grown in 3.0 ml TGY broth to mid-log phase (OD600 of 0.5), treated with CaCl2, and transformed essentially as described previously (12). Competent cells (2 x 108 CFU/ml) were mixed with transforming DNA from strain LS18 (5 µg; 10 µg/ml in the transformation mixture), serially diluted, and plated onto TGY plates to determine total cell numbers and onto TGY plates with streptomycin (5 µg/ml) to assess the number of transformants. Plates were incubated at 30°C and scored after 2 to 3 days. Transformation efficiency was calculated as transformants/µg DNA/total viable cells.

Alternatively, cells were grown to mid-log phase (OD600 of 0.5), harvested, resuspended at 6 x 109 CFU/ml in 100 µl of TGY medium supplemented with 25 mM CaCl2 and 10% glycerol, and frozen at –80°C, as described previously (34). For each experiment, cells were thawed and mixed with transforming DNA (2 µg; 20 µg/ml in the transformation mixture), and transformation was continued as described previously (34). Transformed cells were spread onto plates containing streptomycin (5 or 50 µg/ml) and scored as described above.

Complementation experiments. The D. radiodurans recD gene, along with 948 bp of DNA upstream of the RecD start codon, was PCR amplified from genomic wild-type DNA and ligated into BamHI-cleaved pRAD1 (36) to make pRAD1-recD. The recD gene and upstream DNA inserted into this plasmid were sequenced, and no differences from the sequence in GenBank (accession number AE000513) were found. The pRAD1-recD plasmid was transformed into D. radiodurans cells (wild-type or recD::kan mutant strains) as described above, and transformants were selected for their ability to grow on chloramphenicol and kanamycin. Sensitivity of the recD::kan (pRAD1-recD) cells to UV irradiation was tested as described above.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Verification of recD::kan gene disruption. The presence of the desired recD gene disruption in the recD::kan mutant, and the absence of undisrupted wild-type recD genes, was verified by PCR and Southern blotting. Chromosomal DNA was isolated from wild-type and kanamycin-resistant colonies, and PCRs were done using primers Prom1 and DRecD2, which should anneal to both the wild-type and recD::kan mutant strains (Fig. 1A). The wild-type DNA gave the expected 2.2-kb fragment, while the recD::kan mutant gave the expected 3.2-kb fragment due to the insertion of the kan gene (Fig. 1B). The mutant gave the expected 1.6-kb fragment with primers Prom1 and KanFor, a primer specific for the kan gene, while no fragment was made from wild-type DNA with these primers (Fig. 1B).

The structure of the recD::kan mutant was confirmed by Southern blotting. Genomic DNA from the recD::kan mutant was digested with four different restriction endonucleases and analyzed with a kan gene probe. DNA fragments of the expected size for the desired recD gene disruption were detected for each enzyme used (Fig. 1C). The same membrane was stripped and reprobed with a recD gene probe. The fragments detected were as expected for the disrupted recD gene (and the same as those detected with the kan gene probe), with no detectable undisrupted, wild-type recD gene (Fig. 1C).

Growth of the recD::kan mutant. The recD::kan mutant strain grew at the same rate as the wild type and reached essentially the same final cell density (Fig. 2). Retention of the disrupted gene in the mutant cells, which were grown without kanamycin, was verified by PCR using genomic DNA isolated from the recD::kan cells at the end of the experiment (data not shown).

DNA damage sensitivity of the recD::kan mutant. The recD::kan mutant cells are more sensitive to UV and gamma irradiation than the wild type (Fig. 3A and B). The mutant exhibits a "shoulder" of resistance to moderate doses that is characteristic of wild-type D. radiodurans, but the mutant is more sensitive to higher doses of these types of radiation than the wild type. The recD::kan mutant is also more sensitive to treatment with hydrogen peroxide than the wild type (Fig. 3C). These results are the first indication that any member of the RecD2 protein subfamily (see the introduction) (42) is involved in DNA metabolism in any bacterium in which they have been identified. In contrast to the DNA-damaging agents shown in Fig. 3, recD::kan mutant cells are no more sensitive to MMC and to MMS than are wild type cells (Fig. 4).


Figure 4
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  		FIG. 4. Sensitivity of the recD::kan mutant to MMC and MMS. Closed circles, wild-type strain BAA-816; open inverted triangles, recD mutant. Cells were treated with MMC (10 µg/ml) (A) or with 30 mM MMS (B) for the indicated times and then spread onto plates lacking either drug. The results for MMC are from two independent experiments (n = 6), and the results for MMS are from one experiment (n = 3).

 
UV sensitivity of the {Delta}recD::cam deletion mutant. We attempted to generate a complete deletion of the recD gene starting with wild-type cells but were unsuccessful for unknown reasons. However, we were able to replace the disrupted recD::kan gene with a cam gene and, in the process, delete the entire recD gene sequence (see Materials and Methods). The structure of the {Delta}recD::cam deletion mutation was verified by Southern blotting. A cam gene probe hybridized with chromosomal DNA fragments of the expected size from the deletion mutant, while no fragments were detected with either a kan gene or a recD gene probe (see Fig. S1 in the supplemental material) (data not shown). The {Delta}recD::cam deletion mutant is as sensitive to gamma and UV irradiation as the recD::kan disruption mutant (Fig. 5).


Figure 5
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  		FIG. 5. Sensitivity of the {Delta}recD::cam deletion mutant to gamma and UV irradiation. Irradiation with (A) gamma rays and (B) UV light was done as described in Materials and Methods and the legend of Fig. 3. Closed circles, wild-type strain BAA-816; closed squares, {Delta}recD::cam deletion mutant; open inverted triangles, recD::kan disruption mutant. Results for gamma irradiation are from a single experiment (n = 3), while those for UV irradiation are from two independent experiments (n = 6).

 
Complementation of the recD::kan mutation. We next tested whether the UV sensitivity of the recD::kan mutant strain could be complemented by a plasmid-borne copy of the wild-type recD gene. We inserted the entire recD coding region, as well as a large portion of genomic DNA upstream of recD, into plasmid pRAD1, which replicates at low copy number in D. radiodurans cells. The recD gene in this plasmid should be expressed from its natural promoter, which we presume is present in the upstream DNA. The pRAD1-recD plasmid partially complements the UV sensitivity of the recD::kan mutant (Fig. 6).

We are uncertain why there is reproducibly partial complementation of the recD::kan mutation by plasmid pRAD1-recD. We considered the possibility that the observed phenotype of the recD::kan mutant could be the combined effect of the recD disruption mutation as well as an additional mutation in some gene other than recD. However, we believe that this is very unlikely for the following reasons: (i) the PCR analysis and Southern blots (Fig. 1) indicate that the kan gene insertion is in the recD gene as expected, with no secondary insertion that could contribute to the observed phenotype, and (ii) as described in Materials and Methods, we made the recD::kan disruption mutation three times. Mutants 2 and 3 were equally sensitive to UV and IR, and both were resistant to MMC (data not shown). All three mutant strains showed similar enhanced transformability (see below). These observations make it extremely unlikely that the recD mutant phenotypes could be due to a second mutation elsewhere in the genome, as it is very unlikely that the second mutation would be transferred along with recD::kan to naïve recipient cells.

It is conceivable that the insertion into the recD gene could have a polar effect on neighboring genes. However, an examination of the D. radiodurans genome sequence indicated that there are no other genes in the vicinity of recD that are likely to be involved in DNA metabolism. The nearest annotated open reading frame in the genome downstream of recD begins 229 bp after the predicted recD stop codon. This open reading frame, and the one that follows, would encode proteins of unknown function and with no significant similarity to other proteins in the GenBank database. It is thus very unlikely that the observed phenotype arises from an effect on expression of these putative proteins. The nearest gene upstream of recD is predicted to be transcribed in the opposite direction from recD and thus would be unlikely to feel a polar effect of recD gene disruption.

Transformation efficiency of the recD::kan mutant. Finally, we tested the ability of recD::kan mutant cells to take up and be transformed by exogenous DNA. D. radiodurans is naturally competent to take up DNA from its environment and integrate the transforming DNA into its chromosome via homologous recombination (41). The transformation efficiency of the recD::kan mutant was greater than that of the wild type with transforming DNA from D. radiodurans strain LS18, which carries streptomycin resistance. Using a protocol in which wild-type D. radiodurans is transformed relatively efficiently (12), the recD::kan mutant was transformed with a three- to sevenfold-greater efficiency than the wild type (Table 1). The difference in transformation efficiency between the mutant and wild type was even greater (30- to 100-fold) when cells were treated as described previously (34) (Table 1).


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  		TABLE 1. Transformation efficiency of the recD::kan mutanta

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The RecD protein is well known as a subunit of the RecBCD enzyme and has been studied extensively in that enzyme from E. coli. There has been much less work on the proteins in the RecD2 subfamily that includes homologues from D. radiodurans, B. subtilis, Chlamydia, and other bacteria (see the introduction). The results reported in this paper indicate that RecD serves some function in DNA damage repair and/or recombination, at least in D. radiodurans. A study similar to ours, in which a recD disruption mutation was constructed in the D. radiodurans chromosome, has been reported recently (50). Those workers found, as we did, that the recD mutant is sensitive to treatment with hydrogen peroxide. However, those authors reported that the recD cells are less sensitive to UV than we have found and that they are not sensitive to IR. The reason for this difference between the two sets of results is not clear at this time.

Role of RecD in DNA repair in D. radiodurans. It is not yet possible to specify a precise function for RecD in DNA damage resistance in D. radiodurans. The DNA-damaging agents used in this work give rise to a variety of forms of damage, and the phenotype of the recD mutant is somewhat different from those of other D. radiodurans mutants studied previously, as elaborated below. Thus, much further work is required before RecD can be placed in a specific DNA repair pathway.

A significant effect of gamma irradiation is the production of dsDNA breaks, and a mechanism by which D. radiodurans can repair thousands of double-strand breaks was proposed recently (49). Double-strand ends are thought to be resected by an unknown nuclease (10, 14, 30), and the resulting single-stranded 3' ends serve to prime DNA synthesis either by annealing to homologous single strands or by invading homologous double-stranded regions. D. radiodurans cells with mutations in the recA and polA genes are highly sensitive to IR (9, 19, 20), indicating essential roles for the RecA protein and DNA polymerase I in the repair process. However, the additional enzymes thought to be required for the proposed repair pathway, including helicases that may act at several steps (49), are not yet known.

The sensitivity of the recD mutants to gamma irradiation may indicate a role for RecD in the repair of double-strand breaks. However, the recD mutants are much less sensitive to IR than are the recA and polA mutants. It is possible that RecD does act in double-strand-break repair but that another helicase(s) overlaps in function with RecD and can partially substitute for RecD and thus lessen the effect of the recD mutation. A second possibility is that there is more than one pathway for the repair of DNA damage from IR in D. radiodurans, only one of which is affected by the recD mutation.

The fact that the recD mutant is sensitive to IR and hydrogen peroxide but not to MMC suggests that RecD might be involved in resistance to oxidation damage rather than in the repair of double-strand breaks caused by IR. Repair by nucleotide excision repair of closely spaced single-strand adducts and interstrand cross-links produced by MMC gives rise to double-strand breaks in E. coli (11) and D. radiodurans (25). Consistent with this, D. radiodurans uvrA and uvrD mutants and recA and polA mutants are quite sensitive to MMC as well as to IR. The fact that the recD mutant is not sensitive to MMC thus suggests that it may not act in double-strand-break repair. Both IR and hydrogen peroxide lead to the formation of single-strand breaks and a variety of oxidative products from the DNA bases (15, 23). Double-strand breaks are produced at much lower levels by hydrogen peroxide than by IR, since peroxide-induced DNA damage at adjacent positions in opposite strands is unlikely (15). (The specific effect of H2O2 on the DNA of D. radiodurans, at the concentrations used in our experiments, has not been examined.)

Role for RecD in transformation. The elevated level of transformation in the recD::kan mutant is similar to the effect of mutations in genes encoding several different helicases in other organisms. Interestingly, E. coli recD mutants exhibit three- to sixfold-greater levels of recombination than wild-type cells in some assays (4), and E. coli recD mutants are more readily transformable than the wild type with linear dsDNA (43). The loss of the RecD subunit in E. coli disables the nuclease activity of RecBCD (4), and therefore, transforming DNA is not degraded in the cell. Further work will be needed to see whether RecD is involved in DNA degradation in D. radiodurans. The D. radiodurans recD mutants differ from E. coli recD mutants in that the latter mutants are not sensitive to DNA-damaging agents such as UV light (4, 44).

Several other helicases, including UvrD, its eukaryotic homologue Srs2, and several RecQ homologues, also suppress the levels of homologous recombination. UvrD and Srs2 can displace the RecA protein from DNA to suppress recombination (1, 3, 40, 46), while the RecQ homologues unwind recombination intermediates and prevent their resolution to recombinant products (24, 32, 33). Interestingly, a D. radiodurans uvrD mutant shows lower levels of recombination than does the wild type (37). It could be that RecD serves the antirecombinogenic function in D. radiodurans that is served by the UvrD helicase in E. coli.


   ACKNOWLEDGMENTS
 
We thank John Battista and Michael Daly for gifts of materials and for helpful discussions, Alex Holland and Mary Lidstrom for the gift of E. coli-D. radiodurans shuttle plasmids, Alia Weaver of the University of Maryland gamma irradiation facility for assistance with the 60Co source and gamma irradiation experiments, and Wei Li for assistance with Southern blotting.

This work was supported by funds from the NIH (grant no. GM-39777).


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742. Phone: (301) 405-1821. Fax: (301) 314-9121. E-mail: djulin{at}umd.edu Back

{triangledown} Published ahead of print on 11 May 2007. Back

{dagger} Supplemental material for this article may be found at http://jb.asm.org/. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Journal of Bacteriology, July 2007, p. 5101-5107, Vol. 189, No. 14
0021-9193/07/$08.00+0     doi:10.1128/JB.00409-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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