Identification of the nuclease active site in the multifunctional RecBCD enzyme by creation of a chimeric enzyme.
The recombinational hot spot chi 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 RecB1-929, comprising only the 100 kDa N-terminal domain of RecB, does not. We reported previously that the reconstituted RecB1-929CD 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 an 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 RecBD1080ACD 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.
Yu
M
M
Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA.
Souaya
J
J
Julin
D A
DA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry; D017104:Bacteriophage M13 / Q000737:chemistry; D004265:DNA Helicases / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry; D004706:Endodeoxyribonucleases / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000737:chemistry*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D016415:Sequence Alignment; D013379:Substrate Specificity; D014764:Viral Proteins / Q000737:chemistry
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C060035:gp32 protein, Enterobacteria phage T4; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli; D004265:DNA Helicases