Tetramerization and single-stranded DNA binding properties of native and mutated forms of murine mitochondrial single-stranded DNA-binding proteins

被引:65
作者
Li, K
Williams, RS
机构
[1] UNIV TEXAS,SW MED CTR,DEPT INTERNAL MED,DALLAS,TX 75235
[2] UNIV TEXAS,SW MED CTR,DEPT MOL BIOL,DALLAS,TX 75235
关键词
D O I
10.1074/jbc.272.13.8686
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
We examined previously unexplored aspects of the tetramerization and single-stranded DNA (ssDNA) binding properties of native, precursor, and mutated forms of mitochondrial ssDNA-binding protein (mtSSB) from a mammalian organism (mouse). Tetramic forms of mtSSB reassemble spontaneously after thermal denaturation and undergo subunit exchange. Binding of mtSSB to ssDNA as a function of protein concentration is nonlinear, suggesting a concentration-dependent transition in intrinsic binding affinity and in the topology of the DNA-protein complex. The cleavable presequence at the amino terminus of the precursor form of mtSSB does not disrupt tetramer formation but has a specific inhibitory effect on DNA binding that is not seen in a fusion protein that substitutes a bulkier peptide moiety in this position. Mutated forms of mtSSB bearing amino acid substitutions at highly conserved amino acid positions exhibit subtle or severe defects in ssDNA binding activity and/or tetramerization, even when assembled into heterotetramers in combination with wild-type mtSSB monomers. These experiments provide new insights into structural and functional properties of mammalian mtSSB and have implications for the pathogenesis of human diseases resulting from defects in mtDNA replication.
引用
收藏
页码:8686 / 8694
页数:9
相关论文
共 42 条
[1]  
Ausubel F. M., 1994, CURRENT PROTOCOLS MO
[2]  
BUJALOWSKI W, 1988, J BIOL CHEM, V263, P4629
[3]  
BUJALOWSKI W, 1991, J BIOL CHEM, V266, P1616
[4]   TRYPTOPHAN-54 AND PHENYLALANINE-60 ARE INVOLVED SYNERGISTICALLY IN THE BINDING OF ESCHERICHIA-COLI SSB PROTEIN TO SINGLE-STRANDED POLYNUCLEOTIDES [J].
CASASFINET, JR ;
KHAMIS, MI ;
MAKI, AH ;
CHASE, JW .
FEBS LETTERS, 1987, 220 (02) :347-352
[5]  
CHASE JW, 1986, ANNU REV BIOCHEM, V55, P103, DOI 10.1146/annurev.bi.55.070186.000535
[6]  
CHASE JW, 1985, J BIOL CHEM, V260, P7214
[7]  
CHASE JW, 1984, J BIOL CHEM, V259, P805
[8]   AMINO-ACID 55 PLAYS A CENTRAL ROLE IN TETRAMERIZATION AND FUNCTION OF ESCHERICHIA-COLI SINGLE-STRANDED-DNA BINDING-PROTEIN [J].
CURTH, U ;
BAYER, I ;
GREIPEL, J ;
MAYER, F ;
URBANKE, C ;
MAASS, G .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1991, 196 (01) :87-93
[9]   SINGLE-STRANDED-DNA-BINDING PROTEINS FROM HUMAN MITOCHONDRIA AND ESCHERICHIA-COLI HAVE ANALOGOUS PHYSICOCHEMICAL PROPERTIES [J].
CURTH, U ;
URBANKE, C ;
GREIPEL, J ;
GERBERDING, H ;
TIRANTI, V ;
ZEVIANI, M .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1994, 221 (01) :435-443
[10]   MUTANT SINGLE-STRAND BINDING-PROTEIN OF ESCHERICHIA-COLI - GENETIC AND PHYSIOLOGICAL CHARACTERIZATION [J].
GLASSBERG, J ;
MEYER, RR ;
KORNBERG, A .
JOURNAL OF BACTERIOLOGY, 1979, 140 (01) :14-19