Characterization of a mitochondrially targeted single-stranded DNA-binding protein in Arabidopsis thaliana.
A gene encoding a predicted mitochondrially targeted single-stranded DNA binding protein (mtSSB) was identified in the Arabidopsis thaliana genome sequence. This gene (At4g11060) codes for a protein of 201 amino acids, including a 28-residue putative mitochondrial targeting transit peptide. Protein sequence alignment shows high similarity between the mtSSB protein and single-stranded DNA binding proteins (SSB) from bacteria, including residues conserved for SSB function. Phylogenetic analysis indicates a close relationship between this protein and other mitochondrially targeted SSB proteins. The predicted targeting sequence was fused with the GFP coding region, and the organellar localization of the expressed fusion protein was determined. Specific targeting to mitochondria was observed in in-vitro import experiments and by transient expression of a GFP fusion construct in Arabidopsis leaves after microprojectile bombardment. The mature mtSSB coding region was overexpressed in Escherichia coli and the protein was purified for biochemical characterization. The purified protein binds single-stranded, but not double-stranded, DNA. MtSSB stimulates the homologous strand-exchange activity of E. coli RecA. These results indicate that mtSSB is a functional homologue of the E. coli SSB, and that it may play a role in mitochondrial DNA recombination.
Edmondson
Andrew C
AC
Department of Microbiology and Molecular Biology, Brigham Young University, 775 WIDB, Provo, UT, 84602, USA.
Song
Daqing
D
Alvarez
Luis A
LA
Wall
Melisa K
MK
Almond
David
D
McClellan
David A
DA
Maxwell
Anthony
A
Nielsen
Brent L
BL
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics*; D019470:Biolistics; D019295:Computational Biology; D004268:DNA-Binding Proteins / Q000235:genetics*; D004926:Escherichia coli; D040461:Gene Components; D049452:Green Fluorescent Proteins; D016013:Likelihood Functions; D008928:Mitochondria / Q000378:metabolism*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010802:Phylogeny; D011938:Rec A Recombinases / Q000378:metabolism; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein
D004268:DNA-Binding Proteins; D049452:Green Fluorescent Proteins; D011938:Rec A Recombinases
J Biol Chem. 1987 Jun 25;262(18):8574-83
3298237
Protein Sci. 1999 May;8(5):978-84
10338008
Biochemistry. 1987 Jun 16;26(12):3347-54
3307905
Curr Genet. 1995 Sep;28(4):390-9
8590487
Eur J Biochem. 1989 Feb 1;179(2):399-404
2645140
Mol Genet Genomics. 2003 Jul;269(4):454-63
12768414
Cell. 1989 Jul 14;58(1):69-76
2752423
Plant Mol Biol. 1997 Apr;33(6):1037-50
9154985
Bioinformatics. 2003 Aug 12;19(12):1572-4
12912839
Trends Genet. 1992 Apr;8(4):128-32
1631955
Trends Genet. 1995 Jun;11(6):228-35
7638905
Plant Cell. 1996 Mar;8(3):447-461
12239390
Curr Genet. 2000 May;37(5):304-14
10853767
J Mol Biol. 1996 Feb 2;255(4):564-88
8568898
Nucleic Acids Res. 2000 Jan 1;28(1):155-8
10592209
J Biol Chem. 1987 Aug 15;262(23):10938-45
3301846
Microbiol Rev. 1990 Dec;54(4):342-80
2087220
J Bacteriol. 1987 Aug;169(8):3422-8
3301800
Curr Genet. 1998 Mar;33(3):188-98
9508793
Proc Natl Acad Sci U S A. 1980 Feb;77(2):857-61
6244589
Annu Rev Genet. 1998;32:379-413
9928485
Nucleic Acids Res. 1999 Jan 1;27(1):153-5
9847163
Protein Expr Purif. 2001 Jun;22(1):120-7
11388809
Protein Expr Purif. 1998 Dec;14(3):382-6
9882572
J Mol Biol. 1998 Mar 6;276(4):745-58
9500926
J Biol Chem. 1980 Apr 10;255(7):2897-901
6244299
Curr Genet. 1998 Oct;34(4):303-12
9799364
Arch Biochem Biophys. 2001 Apr 1;388(1):165-70
11361133
Proc Natl Acad Sci U S A. 1981 Jul;78(7):4274-8
6270666
Proc Natl Acad Sci U S A. 2004 May 18;101(20):7821-6
15136745
FEBS Lett. 1987 Aug 17;220(2):347-52
3301414
Eur J Biochem. 1999 Apr;261(2):361-70
10215845
J Biol Chem. 1983 Mar 10;258(5):3346-55
6298232
Trends Biochem Sci. 1999 Jan;24(1):34-6
10087920
Biol Chem. 1997 Aug;378(8):859-62
9377482
Methods Enzymol. 1983;97:476-84
6361481
Annu Rev Biochem. 1994;63:527-70
7979247
Mol Gen Genet. 1986 Mar;202(3):421-8
3520238
Plant Mol Biol. 1995 Oct;29(2):245-54
7579176
Adv Protein Chem. 2000;54:277-344
10829231
J Biol Chem. 2001 Dec 7;276(49):45959-68
11577073