Identification of the nuclease active site in the multifunctional RecBCD enzyme by creation of a chimeric enzyme

被引:81
作者
Yu, M [1 ]
Souaya, J [1 ]
Julin, DA [1 ]
机构
[1] Univ Maryland, Dept Chem & Biochem, College Pk, MD 20742 USA
关键词
RecBCD; endonuclease; gene; 32; protein; recombination; Chi sequence;
D O I
10.1006/jmbi.1998.2127
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
The recombinational hot spot :a modulates the nuclease and helicase activities of the RecBCD enzyme, leading to generation of an early DNA intermediate for homologous recombination. Here we identify the subunit location of the nuclease active site in RecBCD. The isolated RecB protein cleaves circular single-stranded M13 phage DNA, but RecB(1-929), comprising only the 100 kDa N-terminal domain of RecB, does not. We reported previously that the reconstituted RecB(1-929)CD enzyme also is not a nuclease, suggesting that the C-terminal 30 kDa domain of RecB is a non-specific ssDNA endonuclease. However, we were unable to detect nuclease activity with the subtilisin-generated C-terminal 30 kDa fragment of RecB. Since the subtilisin-generated fragment did not bind to a ssDNA-agarose column, we designed a chimeric enzyme by attaching the C-terminal 30 kDa domain of RecB to the gene 32 protein of T4 phage, a ssDNA binding protein that does not have strand scission ability. In addition, Asp427 in the chimeric enzyme (Asp1080 in RecB), a residue that is conserved among several RecB homologs, was substituted to alanine (the D427A mutant). The wild-type chimeric enzyme cleaves the M13 DNA and the D427A mutation abolishes the endonuclease activity of the chimeric enzyme but does not affect its DNA binding ability. This finding indicates aln unusual bipartite nature in the structural organization of RecB, in which the DNA-binding function is located in the N-terminal 100 kDa domain and the nuclease catalytic domain is located in the C-terminal 30 kDa domain. The purified RecB(D1080A)CD mutant is a processive helicase but not a nuclease, demonstrating that RecBCD has a single nuclease active site in the C-terminal 30 kDa domain of RecB. (C) 1998 Academic Press.
引用
收藏
页码:797 / 808
页数:12
相关论文
共 59 条
[1]  
AMUNDSEN SK, 1990, GENETICS, V126, P25
[2]   The translocating RecBCD enzyme stimulates recombination by directing RecA protein onto ssDNA in a chi-regulated manner [J].
Anderson, DG ;
Kowalczykowski, SC .
CELL, 1997, 90 (01) :77-86
[3]   Chi-activated RecBCD enzyme possesses 5'->3' nucleolytic activity, but RecBC enzyme does not: Evidence suggesting that the alteration induced by Chi is not simply ejection of the RecD subunit [J].
Anderson, DG ;
Churchill, JJ ;
Kowalczykowski, SC .
GENES TO CELLS, 1997, 2 (02) :117-128
[4]   The reduced levels of χ recognition exhibited by the RecBC1004D enzyme reflect its recombination defect in vivo [J].
Arnold, DA ;
Bianco, PR ;
Kowalczykowski, SC .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1998, 273 (26) :16476-16486
[5]   STRUCTURAL BASIS FOR THE 3'-5' EXONUCLEASE ACTIVITY OF ESCHERICHIA-COLI DNA-POLYMERASE-I - A 2 METAL-ION MECHANISM [J].
BEESE, LS ;
STEITZ, TA .
EMBO JOURNAL, 1991, 10 (01) :25-33
[6]   The recombination hotspot Chi is recognized by the translocating RecBCD enzyme as the single strand of DNA containing the sequence 5'-GCTGGTGG-3' [J].
Bianco, PR ;
Kowalczykowski, SC .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1997, 94 (13) :6706-6711
[7]   ESCHERICHIA-COLI RECBCD ENZYME - INDUCIBLE OVERPRODUCTION AND RECONSTITUTION OF THE ATP-DEPENDENT DEOXYRIBONUCLEASE FROM PURIFIED SUBUNITS [J].
BOEHMER, PE ;
EMMERSON, PT .
GENE, 1991, 102 (01) :1-6
[8]   COMPARATIVE DNA-SEQUENCE FEATURES IN 2 LONG ESCHERICHIA-COLI CONTIGS [J].
CARDON, LR ;
BURGE, C ;
SCHACHTEL, GA ;
BLAISDELL, BE ;
KARLIN, S .
NUCLEIC ACIDS RESEARCH, 1993, 21 (16) :3875-3884
[9]  
CHASE JW, 1986, ANNU REV BIOCHEM, V55, P103, DOI 10.1146/annurev.bi.55.070186.000535
[10]   ESCHERICHIA-COLI RECBC DELETION MUTANTS [J].
CHAUDHURY, AM ;
SMITH, GR .
JOURNAL OF BACTERIOLOGY, 1984, 160 (02) :788-791