A Major Role of the RecFOR Pathway in DNA Double-Strand-Break Repair through ESDSA in
In Deinococcus radiodurans, the extreme resistance to DNA–shattering treatments such as ionizing radiation or desiccation is correlated with its ability to reconstruct a functional genome from hundreds of chromosomal fragments. The rapid reconstitution of an intact genome is thought to occur through an extended synthesis-dependent strand annealing process (ESDSA) followed by DNA recombination. Here, we investigated the role of key components of the RecF pathway in ESDSA in this organism naturally devoid of RecB and RecC proteins. We demonstrate that inactivation of RecJ exonuclease results in cell lethality, indicating that this protein plays a key role in genome maintenance. Cells devoid of RecF, RecO, or RecR proteins also display greatly impaired growth and an important lethal sectoring as bacteria devoid of RecA protein. Other aspects of the phenotype of recFOR knock-out mutants paralleled that of a ΔrecA mutant: ΔrecFOR mutants are extremely radiosensitive and show a slow assembly of radiation-induced chromosomal fragments, not accompanied by DNA synthesis, and reduced DNA degradation. Cells devoid of RecQ, the major helicase implicated in repair through the RecF pathway in E. coli, are resistant to γ-irradiation and have a wild-type DNA repair capacity as also shown for cells devoid of the RecD helicase; in contrast, ΔuvrD mutants show a markedly decreased radioresistance, an increased latent period in the kinetics of DNA double-strand-break repair, and a slow rate of fragment assembly correlated with a slow rate of DNA synthesis. Combining RecQ or RecD deficiency with UvrD deficiency did not significantly accentuate the phenotype of ΔuvrD mutants. In conclusion, RecFOR proteins are essential for DNA double-strand-break repair through ESDSA whereas RecJ protein is essential for cell viability and UvrD helicase might be involved in the processing of double stranded DNA ends and/or in the DNA synthesis step of ESDSA.
Vyšlo v časopise:
A Major Role of the RecFOR Pathway in DNA Double-Strand-Break Repair through ESDSA in. PLoS Genet 6(1): e32767. doi:10.1371/journal.pgen.1000774
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.pgen.1000774
Souhrn
In Deinococcus radiodurans, the extreme resistance to DNA–shattering treatments such as ionizing radiation or desiccation is correlated with its ability to reconstruct a functional genome from hundreds of chromosomal fragments. The rapid reconstitution of an intact genome is thought to occur through an extended synthesis-dependent strand annealing process (ESDSA) followed by DNA recombination. Here, we investigated the role of key components of the RecF pathway in ESDSA in this organism naturally devoid of RecB and RecC proteins. We demonstrate that inactivation of RecJ exonuclease results in cell lethality, indicating that this protein plays a key role in genome maintenance. Cells devoid of RecF, RecO, or RecR proteins also display greatly impaired growth and an important lethal sectoring as bacteria devoid of RecA protein. Other aspects of the phenotype of recFOR knock-out mutants paralleled that of a ΔrecA mutant: ΔrecFOR mutants are extremely radiosensitive and show a slow assembly of radiation-induced chromosomal fragments, not accompanied by DNA synthesis, and reduced DNA degradation. Cells devoid of RecQ, the major helicase implicated in repair through the RecF pathway in E. coli, are resistant to γ-irradiation and have a wild-type DNA repair capacity as also shown for cells devoid of the RecD helicase; in contrast, ΔuvrD mutants show a markedly decreased radioresistance, an increased latent period in the kinetics of DNA double-strand-break repair, and a slow rate of fragment assembly correlated with a slow rate of DNA synthesis. Combining RecQ or RecD deficiency with UvrD deficiency did not significantly accentuate the phenotype of ΔuvrD mutants. In conclusion, RecFOR proteins are essential for DNA double-strand-break repair through ESDSA whereas RecJ protein is essential for cell viability and UvrD helicase might be involved in the processing of double stranded DNA ends and/or in the DNA synthesis step of ESDSA.
Zdroje
1. ZahradkaK
SladeD
BailoneA
SommerS
AverbeckD
2006 Reassembly of shattered chromosomes in Deinococcus radiodurans. Nature 443 569 573
2. SladeD
LindnerAB
PaulG
RadmanM
2009 Recombination and replication in DNA repair of heavily irradiated Deinococcus radiodurans. Cell 136 1044 1055
3. SmithGR
1989 Homologous recombination in prokaryotes: enzymes and controlling sites. Genome 31 520 527
4. KuzminovA
1999 Recombinational repair of DNA damage in Escherichia coli and bacteriophage lambda. Microbiol Mol Biol Rev 63 751 813
5. KowalczykowskiSC
2000 Initiation of genetic recombination and recombination-dependent replication. Trends Biochem Sci 25 156 165
6. HoriiZ
ClarkAJ
1973 Genetic analysis of the recF pathway to genetic recombination in Escherichia coli K12: isolation and characterization of mutants. J Mol Biol 80 327 344
7. ClarkAJ
SandlerSJ
WillisDK
ChuCC
BlanarMA
1984 Genes of the RecE and RecF pathways of conjugational recombination in Escherichia coli. Cold Spring Harb Symp Quant Biol 49 453 462
8. NakayamaH
NakayamaK
NakayamaR
IrinoN
NakayamaY
1984 Isolation and genetic characterization of a thymineless death-resistant mutant of Escherichia coli K12: identification of a new mutation (recQ1) that blocks the RecF recombination pathway. Mol Gen Genet 195 474 480
9. KolodnerR
FishelRA
HowardM
1985 Genetic recombination of bacterial plasmid DNA: effect of RecF pathway mutations on plasmid recombination in Escherichia coli. J Bacteriol 163 1060 1066
10. MahdiAA
LloydRG
1989 Identification of the recR locus of Escherichia coli K-12 and analysis of its role in recombination and DNA repair. Mol Gen Genet 216 503 510
11. LloydRG
EvansNP
BuckmanC
1987 Formation of recombinant lacZ+ DNA in conjugational crosses with a recB mutant of Escherichia coli K12 depends on recF, recJ, and recO. Mol Gen Genet 209 135 141
12. CromieGA
2009 Phylogenetic ubiquity and shuffling of the bacterial RecBCD and AddAB recombination complexes. J Bacteriol 191 5076 5084
13. RochaEP
CornetE
MichelB
2005 Comparative and evolutionary analysis of the bacterial homologous recombination systems. PLoS Genet 1 e15 doi:10.1371/journal.pgen.0010015
14. WangJ
JulinDA
2004 DNA helicase activity of the RecD protein from Deinococcus radiodurans. J Biol Chem 279 52024 52032
15. ZhouQ
ZhangX
XuH
XuB
HuaY
2007 A new role of Deinococcus radiodurans RecD in antioxidant pathway. FEMS Microbiol Lett 271 118 125
16. ServinskyMD
JulinDA
2007 Effect of a recD mutation on DNA damage resistance and transformation in Deinococcus radiodurans. J Bacteriol 189 5101 5107
17. MisraHS
KhairnarNP
KotaS
ShrivastavaS
JoshiVP
2006 An exonuclease I-sensitive DNA repair pathway in Deinococcus radiodurans: a major determinant of radiation resistance. Mol Microbiol 59 1308 1316
18. MennecierS
CosteG
ServantP
BailoneA
SommerS
2004 Mismatch repair ensures fidelity of replication and recombination in the radioresistant organism Deinococcus radiodurans. Mol Genet Genomics 272 460 469
19. HansenMT
1978 Multiplicity of genome equivalents in the radiation-resistant bacterium Micrococcus radiodurans. J Bacteriol 134 71 75
20. Harsojo
KitayamaS
MatsuyamaA
1981 Genome multiplicity and radiation resistance in Micrococcus radiodurans. J Biochem (Tokyo) 90 877 880
21. NguyenHH
Bouthier de la TourC
ToueilleM
VannierF
SommerS
2009 The essential histone-like protein HU plays a major role in Deinococcus radiodurans nucleoid compaction. Mol Microbiol 73 240 252
22. WhiteO
EisenJA
HeidelbergJF
HickeyEK
PetersonJD
1999 Genome sequence of the radioresistant bacterium Deinococcus radiodurans R1. Science 286 1571 1577
23. MendoncaVM
KlepinHD
MatsonSW
1995 DNA helicases in recombination and repair: construction of a delta uvrD delta helD delta recQ mutant deficient in recombination and repair. J Bacteriol 177 1326 1335
24. BattistaJR
1997 Against all odds: the survival strategies of Deinococcus radiodurans. Annu Rev Microbiol 51 203 224
25. KikuchiMNI
KitayamaS
WatanabeH
YamamotoK
1999 Genomic organization of the radioresistant bacterium Deinococcus radiodurans : physical map and evidence for multiple replicons. FEMS Microbiol Lett 174 151 157
26. Bonacossa de AlmeidaC
CosteG
SommerS
BailoneA
2002 Quantification of RecA protein in Deinococcus radiodurans reveals involvement of RecA, but not LexA, in its regulation. Mol Genet Genomics 268 28 41
27. SatohK
NarumiI
KikuchiM
KitayamaS
YanagisawaT
2002 Characterization of RecA424 and RecA670 proteins from Deinococcus radiodurans. J Biochem (Tokyo) 131 121 129
28. ShanQ
BorkJM
WebbBL
InmanRB
CoxMM
1997 RecA protein filaments: end-dependent dissociation from ssDNA and stabilization by RecO and RecR proteins. J Mol Biol 265 519 540
29. WebbBL
CoxMM
InmanRB
1997 Recombinational DNA repair: the RecF and RecR proteins limit the extension of RecA filaments beyond single-strand DNA gaps. Cell 91 347 356
30. BorkJM
CoxMM
InmanRB
2001 The RecOR proteins modulate RecA protein function at 5′ ends of single-stranded DNA. EMBO J 20 7313 7322
31. MorimatsuK
KowalczykowskiSC
2003 RecFOR proteins load RecA protein onto gapped DNA to accelerate DNA strand exchange: a universal step of recombinational repair. Mol Cell 11 1337 1347
32. InoueJ
HondaM
IkawaS
ShibataT
MikawaT
2008 The process of displacing the single-stranded DNA-binding protein from single-stranded DNA by RecO and RecR proteins. Nucleic Acids Res 36 94 109
33. SakaiA
CoxMM
2009 RecFOR and RecOR as distinct RecA loading pathways. J Biol Chem 284 3264 3272
34. LeeBI
KimKH
ShimSM
HaKS
YangJK
2004 Crystallization and preliminary X-ray crystallographic analysis of the RecR protein from Deinococcus radiodurans, a member of the RecFOR DNA-repair pathway. Acta Crystallogr D Biol Crystallogr 60 379 381
35. LeirosI
TimminsJ
HallDR
McSweeneyS
2005 Crystal structure and DNA-binding analysis of RecO from Deinococcus radiodurans. EMBO J 24 906 918
36. KorolevaO
MakharashviliN
CourcelleCT
CourcelleJ
KorolevS
2007 Structural conservation of RecF and Rad50: implications for DNA recognition and RecF function. EMBO J 26 867 877
37. MakharashviliN
MiT
KorolevaO
KorolevS
2009 RecR-mediated modulation of RecF dimer specificity for single- and double-stranded DNA. J Biol Chem 284 1425 1434
38. LovettST
ClarkAJ
1984 Genetic analysis of the recJ gene of Escherichia coli K-12. J Bacteriol 157 190 196
39. GarzonA
BeuzonCR
MahanMJ
CasadesusJ
1996 recB recJ mutants of Salmonella typhimurium are deficient in transductional recombination, DNA repair and plasmid maintenance. Mol Gen Genet 250 570 580
40. KicksteinE
HarmsK
WackernagelW
2007 Deletions of recBCD or recD influence genetic transformation differently and are lethal together with a recJ deletion in Acinetobacter baylyi. Microbiology 153 2259 2270
41. CourcelleJ
DonaldsonJR
ChowKH
CourcelleCT
2003 DNA damage-induced replication fork regression and processing in Escherichia coli. Science 299 1064 1067
42. CourcelleCT
ChowKH
CaseyA
CourcelleJ
2006 Nascent DNA processing by RecJ favors lesion repair over translesion synthesis at arrested replication forks in Escherichia coli. Proc Natl Acad Sci U S A 103 9154 9159
43. CoxMM
GoodmanMF
KreuzerKN
SherrattDJ
SandlerSJ
2000 The importance of repairing stalled replication forks. Nature 404 37 41
44. PenningtonJM
RosenbergSM
2007 Spontaneous DNA breakage in single living Escherichia coli cells. Nat Genet 39 797 802
45. KilloranMP
KeckJL
2006 Three HRDC domains differentially modulate Deinococcus radiodurans RecQ DNA helicase biochemical activity. J Biol Chem 281 12849 12857
46. HuangL
HuaX
LuH
GaoG
TianB
2007 Three tandem HRDC domains have synergistic effect on the RecQ functions in Deinococcus radiodurans. DNA Repair (Amst) 6 167 176
47. ChenH
HuangL
HuaX
YingL
HuY
2009 Pleiotropic Effects of RecQ in Deinococcus radiodurans. Genomics
48. MatsonSW
RobertsonAB
2006 The UvrD helicase and its modulation by the mismatch repair protein MutL. Nucleic Acids Res 34 4089 4097
49. MendoncaVM
Kaiser-RogersK
MatsonSW
1993 Double helicase II (uvrD)-helicase IV (helD) deletion mutants are defective in the recombination pathways of Escherichia coli. J Bacteriol 175 4641 4651
50. CaoZ
JulinDA
2009 Characterization in vitro and in vivo of the DNA helicase encoded by Deinococcus radiodurans locus DR1572. DNA Repair (Amst) 8 612 619
51. KimJI
SharmaAK
AbbottSN
WoodEA
DwyerDW
2002 RecA Protein from the extremely radioresistant bacterium Deinococcus radiodurans: expression, purification, and characterization. J Bacteriol 184 1649 1660
52. MorelP
HejnaJA
EhrlichSD
CassutoE
1993 Antipairing and strand transferase activities of E. coli helicase II (UvrD). Nucleic Acids Res 21 3205 3209
53. VeauteX
DelmasS
SelvaM
JeussetJ
Le CamE
2005 UvrD helicase, unlike Rep helicase, dismantles RecA nucleoprotein filaments in Escherichia coli. EMBO J 24 180 189
54. LestiniR
MichelB
2007 UvrD controls the access of recombination proteins to blocked replication forks. EMBO J 26 3804 3814
55. OssannaN
MountDW
1989 Mutations in uvrD induce the SOS response in Escherichia coli. J Bacteriol 171 303 307
56. KlinkertMQ
KleinA
Abdel-MonemM
1980 Studies on the functions of DNA helicase I and DNA helicase II of Escherichia coli. J Biol Chem 255 9746 9752
57. KuhnB
Abdel-MonemM
1982 DNA synthesis at a fork in the presence of DNA helicases. Eur J Biochem 125 63 68
58. LahueRS
AuKG
ModrichP
1989 DNA mismatch correction in a defined system. Science 245 160 164
59. BruandC
EhrlichSD
2000 UvrD-dependent replication of rolling-circle plasmids in Escherichia coli. Mol Microbiol 35 204 210
60. HarrisDR
TanakaM
SavelievSV
JolivetE
EarlAM
2004 Preserving genome integrity: the DdrA protein of Deinococcus radiodurans R1. PLoS Biol 2 e304 doi:10.1371/journal.pbio.0020304
61. NoraisCA
Chitteni-PattuS
WoodEA
InmanRB
CoxMM
2009 DdrB protein, an alternative Deinococcus radiodurans SSB induced by ionizing radiation. J Biol Chem 284 21402 21411
62. TanakaM
EarlAM
HowellHA
ParkMJ
EisenJA
2004 Analysis of Deinococcus radiodurans's transcriptional response to ionizing radiation and desiccation reveals novel proteins that contribute to extreme radioresistance. Genetics 168 21 33
63. JolivetE
LecointeF
CosteG
SatohK
NarumiI
2006 Limited concentration of RecA delays DNA double-strand break repair in Deinococcus radiodurans R1. Mol Microbiol 59 338 349
64. Levin-ZaidmanS
EnglanderJ
ShimoniE
SharmaAK
MintonKW
2003 Ringlike structure of the Deinococcus radiodurans genome: a key to radioresistance? Science 299 254 256
65. ZimmermanJM
BattistaJR
2005 A ring-like nucleoid is not necessary for radioresistance in the Deinococcaceae. BMC Microbiol 5 17
66. MeimaR
RothfussHM
GewinL
LidstromME
2001 Promoter cloning in the radioresistant bacterium Deinococcus radiodurans. J Bacteriol 183 3169 3175
67. MennecierS
ServantP
CosteG
BailoneA
SommerS
2006 Mutagenesis via IS transposition in Deinococcus radiodurans. Mol Microbiol 59 317 325
68. BentchikouE
ServantP
CosteG
SommerS
2007 Additive effects of SbcCD and PolX deficiencies in the in vivo repair of DNA double-strand breaks in Deinococcus radiodurans. J Bacteriol 189 4784 4790
69. LecointeF
ShevelevIV
BailoneA
SommerS
HubscherU
2004 Involvement of an X family DNA polymerase in double-stranded break repair in the radioresistant organism Deinococcus radiodurans. Mol Microbiol 53 1721 1730
70. MoseleyBE
CoplandHF
1978 Four mutants of Micrococcus radiodurans defective in the ability to repair DNA damaged by mitomycin-C, two of which have wild-type resistance to ultraviolet radiation. Mol Gen Genet 160 331 337
71. EarlAM
RankinSK
KimKP
LamendolaON
BattistaJR
2002 Genetic evidence that the uvsE gene product of Deinococcus radiodurans R1 is a UV damage endonuclease. J Bacteriol 184 1003 1009
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