Characterization of a Single-Stranded DNA-Binding Protein from Pseudomonas aeruginosa PAO1

被引：20

作者：

Jan, Hau-Chern ^{[1
]}

Lee, Yen-Ling ^{[1
]}

Huang, Cheng-Yang ^{[1
,2
]}

机构：

[1] Chung Shan Med Univ, Dept Biomed Sci, Taichung, Taiwan

[2] Chung Shan Med Univ Hosp, Dept Med Res, Taichung, Taiwan

来源：

PROTEIN JOURNAL | 2011年 / 30卷 / 01期

关键词：

SSB; DNA replication; EMSA; ssDNA binding mode; ESCHERICHIA-COLI SSB; NUCLEIC-ACID BINDING; CRYSTAL-STRUCTURE; REPLICATION PROTEIN; THERMUS-AQUATICUS; SULFOLOBUS-SOLFATARICUS; TERMINAL DOMAIN; IDENTIFICATION; SSDNA; MODES;

D O I：

10.1007/s10930-010-9297-6

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

Single-stranded DNA-binding protein (SSB) plays an important role in DNA metabolism, such as in DNA replication, repair, and recombination, and is essential for cell survival. We characterized the single-stranded DNA (ssDNA)-binding properties of Pseudomonas aeruginosa PAO1 SSB (PaSSB) by using fluorescence quenching measurements and electrophoretic mobility shift analysis (EMSA). Analysis of purified PaSSB by gel filtration chromatography revealed a stable tetramer in solution. In fluorescence titrations, PaSSB bound 22-32 nucleotides (nt) per tetramer depending on salt concentration. Using EMSA, we characterized the stoichiometry of PaSSB complexed with a series of ssDNA homopolymers, and the size of the binding site was determined to be 29 +/- A 1 nt. Furthermore, EMSA results indicated that the dissociation constants of PaSSB for the first tetramer were less than those for the second tetramer. On the basis of these biophysical analyses, the ssDNA binding mode of PaSSB is expected to be noncooperative.

引用

页码：20 / 26

页数：7

共 43 条

[1] STRUCTURAL BASIS FOR THE NUCLEIC-ACID BINDING COOPERATIVITY OF BACTERIOPHAGE-T4 GENE-32 PROTEIN - THE (LYS/ARG)3(SER/THR)2 (LAST) MOTIF
CASASFINET, JR
FISCHER, KR
KARPEL, RL
[J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1992, 89 (03) : 1050 - 1054
[2] Single-Stranded DNA-Binding Protein Complex from Helicobacter pylori Suggests an ssDNA-Binding Surface
Chan, Kun-Wei
Lee, Yi-Juan
Wang, Chia-Hung
Huang, Haimei
Sun, Yuh-Ju
[J]. JOURNAL OF MOLECULAR BIOLOGY, 2009, 388 (03) : 508 - 519
[3] CHASE JW, 1986, ANNU REV BIOCHEM, V55, P103, DOI 10.1146/annurev.bi.55.070186.000535
[4] In vitro and in vivo function of the C-terminus of Escherichia coli single-stranded DNA binding protein
Curth, U
Genschel, J
Urbanke, C
Greipel, J
[J]. NUCLEIC ACIDS RESEARCH, 1996, 24 (14) : 2706 - 2711
[5] MULTIPLE BINDING MODES OF THE SINGLE-STRANDED-DNA BINDING-PROTEIN FROM ESCHERICHIA-COLI AS DETECTED BY TRYPTOPHAN FLUORESCENCE AND SITE-DIRECTED MUTAGENESIS
CURTH, U
GREIPEL, J
URBANKE, C
MAASS, G
[J]. BIOCHEMISTRY, 1993, 32 (10) : 2585 - 2591
[6] Dabrowski S, 2002, MICROBIOL-SGM, V148, P3307, DOI 10.1099/00221287-148-10-3307
[7] Fairall L, 2000, PRACT APPROACH SER, P65
[8] 3D structure of Thermus aquaticus single-stranded DNA-binding protein gives insight into the functioning of SSB proteins
Fedorov, Roman
Witte, Gregor
Urbanke, Claus
Manstein, Dietmar J.
Curth, Ute
[J]. NUCLEIC ACIDS RESEARCH, 2006, 34 (22) : 6708 - 6717
[9] Isolation, sequencing and overproduction of the single-stranded DNA binding protein from Pseudomonas aeruginosa PAO
Genschel, J
Litz, L
Hubert, T
Roemling, U
Urbanke, C
[J]. GENE, 1996, 182 (1-2) : 137 - 143
[10] A distinctive single-stranded DNA-binding protein from the Archaeon Sulfolobus solfataricus
Haseltine, CA
Kowalczykowski, SC
[J]. MOLECULAR MICROBIOLOGY, 2002, 43 (06) : 1505 - 1515

← 1 2 3 4 5 →