DNA strand exchange promoted by RecA K72R - Two reaction phases with different Mg2+ requirements

被引:115
作者
Shan, Q [1 ]
Cox, MM [1 ]
Inman, RB [1 ]
机构
[1] UNIV WISCONSIN,DEPT BIOCHEM,MADISON,WI 53706
关键词
D O I
10.1074/jbc.271.10.5712
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
Replacement of lysine 72 in RecA protein with arginine produces a mutant protein that binds but does not hydrolyze ATP. The protein nevertheless promotes DNA strand exchange (Rehrauer, W. M., and Kowalczykowski, S. C. (1993) J. Biol. Chem. 268, 1292-1297). With RecA K72R protein, the formation of the hybrid DNA product of strand exchange is greatly affected by the concentration of Mg2+ in ways that reflect the concentration of a Mg . dATP complex. When Mg2+ is present at concentrations just sufficient to form the Mg . dATP complex, substantial generation of completed product hybrid DNAs over 7 kilobase pairs in length is observed (albeit slowly). Higher levels of Mg2+ are required for optimal uptake of substrate duplex DNA into the nucleoprotein filament, indicating that the formation of joint molecules is facilitated by Mg2+ levels that inhibit the subsequent migration of a DNA branch. We also show that the strand exchange reaction promoted by RecA K72R, regardless of the Mg2+ concentration, is bidirectional and incapable of bypassing structural barriers in the DNA or accommodating four DNA strands. The reaction exhibits the same limitations as that promoted by wild type RecA protein in the presence of adenosine 5'-O-(3-thio)triphosphate. The Mg2+ effects, the limitations of RecA-mediated DNA strand exchange in the absence of ATP hydrolysis, and unusual DNA structures observed by electron microscopy in some experiments, are interpreted in the context of a model in which a fast phase of DNA strand exchange produces a discontinuous three-stranded DNA pairing intermediate, followed by a slow phase in which the discontinuities are resolved. The mutant protein also facilitates the autocatalytic cleavage of the LexA repressor, but at a reduced rate.
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页码:5712 / 5724
页数:13
相关论文
共 51 条
[1]   STABLE SYNAPSIS OF HOMOLOGOUS DNA-MOLECULES MEDIATED BY THE ESCHERICHIA-COLI RECA PROTEIN INVOLVES LOCAL EXCHANGE OF DNA STRANDS [J].
ADZUMA, K .
GENES & DEVELOPMENT, 1992, 6 (09) :1679-1694
[2]  
ALBERTY RA, 1969, J BIOL CHEM, V244, P3290
[3]  
[Anonymous], 1980, ADV BACTERIAL GENET
[4]   Evidence for the coupling of ATP hydrolysis to the final (extension) phase of RecA protein-mediated DNA strand exchange [J].
Bedale, WA ;
Cox, MM .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1996, 271 (10) :5725-5732
[5]  
BRENNER SL, 1987, J BIOL CHEM, V262, P4011
[6]   HOMOLOGOUS GENETIC-RECOMBINATION - THE PIECES BEGIN TO FALL INTO PLACE [J].
CLARK, AJ ;
SANDLER, SJ .
CRITICAL REVIEWS IN MICROBIOLOGY, 1994, 20 (02) :125-142
[8]  
COX MM, 1981, J BIOL CHEM, V256, P4676
[9]   WHY DOES RECA PROTEIN HYDROLYZE ATP [J].
COX, MM .
TRENDS IN BIOCHEMICAL SCIENCES, 1994, 19 (05) :217-222
[10]   RELATING BIOCHEMISTRY TO BIOLOGY - HOW THE RECOMBINATIONAL REPAIR FUNCTION OF RECA PROTEIN IS MANIFESTED IN ITS MOLECULAR-PROPERTIES [J].
COX, MM .
BIOESSAYS, 1993, 15 (09) :617-623