Structure of RecJ Exonuclease Defines Its Specificity for Single-stranded DNA*

RecJ is a single-stranded DNA (ssDNA)-specific 5′-3′ exonuclease that plays an important role in DNA repair and recombination. To elucidate how RecJ achieves its high specificity for ssDNA, we determined the entire structures of RecJ both in a ligand-free form and in a complex with Mn2+ or Mg2+ by x-ray crystallography. The entire RecJ consists of four domains that form a molecule with an O-like structure. One of two newly identified domains had structural similarities to an oligonucleotide/oligosaccharide-binding (OB) fold. The OB fold domain alone could bind to DNA, indicating that this domain is a novel member of the OB fold superfamily. The truncated RecJ containing only the core domain exhibited much lower affinity for the ssDNA substrate compared with intact RecJ. These results support the hypothesis that these structural features allow specific binding of RecJ to ssDNA. In addition, the structure of the RecJ-Mn2+ complex suggests that the hydrolysis reaction catalyzed by RecJ proceeds through a two-metal ion mechanism.

[1]  A. Murzin.  OB(oligonucleotide/oligosaccharide binding)‐fold: common structural and functional solution for non‐homologous sequences. , 1993, The EMBO journal.

[2]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[3]  E. Bochkareva,et al.  From RPA to BRCA2: lessons from single-stranded DNA binding by the OB-fold. , 2004, Current opinion in structural biology.

[4]  D. Rao,et al.  Orchestration of Haemophilus influenzae RecJ exonuclease by interaction with single-stranded DNA-binding protein. , 2009, Journal of molecular biology.

[5]  T. Steitz,et al.  Structural basis for the 3′‐5′ exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism. , 1991, The EMBO journal.

[6]  R. Masui,et al.  Overexpression, purification and characterization of RecJ protein from Thermus thermophilus HB8 and its core domain. , 2001, Nucleic acids research.

[7]  Jack Snoeyink,et al.  Nucleic Acids Research Advance Access published April 22, 2007 MolProbity: all-atom contacts and structure validation for proteins and nucleic acids , 2007 .

[8]  S. Lovett,et al.  Redundant Exonuclease Involvement in Escherichia coli Methyl-directed Mismatch Repair* , 2001, The Journal of Biological Chemistry.

[9]  G. Dianov,et al.  Release of 5'-terminal deoxyribose-phosphate residues from incised abasic sites in DNA by the Escherichia coli RecJ protein. , 1994, Nucleic acids research.

[10]  Deborah S Wuttke,et al.  Nucleic acid recognition by OB-fold proteins. , 2003, Annual review of biophysics and biomolecular structure.

[11]  D. Hall,et al.  Crystal structure and DNA‐binding analysis of RecO from Deinococcus radiodurans , 2005, The EMBO journal.

[12]  S. Lovett,et al.  A Thermostable Single-Strand DNase fromMethanococcus jannaschii Related to the RecJ Recombination and Repair Exonuclease from Escherichia coli , 2000, Journal of bacteriology.

[13]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[14]  S. Lovett,et al.  In vivo requirement for RecJ, ExoVII, ExoI, and ExoX in methyl-directed mismatch repair , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[15]  Wei Yang,et al.  Crystal Structures of RNase H Bound to an RNA/DNA Hybrid: Substrate Specificity and Metal-Dependent Catalysis , 2005, Cell.

[16]  A. Vagin,et al.  MOLREP: an Automated Program for Molecular Replacement , 1997 .

[17]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[18]  S. White,et al.  The crystal structure of the cytosolic exopolyphosphatase from Saccharomyces cerevisiae reveals the basis for substrate specificity. , 2007, Journal of molecular biology.

[19]  E. Koonin,et al.  A novel family of predicted phosphoesterases includes Drosophila prune protein and bacterial RecJ exonuclease. , 1998, Trends in biochemical sciences.

[20]  Identification and purification of a single-stranded-DNA-specific exonuclease encoded by the recJ gene of Escherichia coli. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[21]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[22]  S. Lovett,et al.  RecJ exonuclease: substrates, products and interaction with SSB , 2006, Nucleic acids research.

[23]  Randy J Read,et al.  Electronic Reprint Biological Crystallography Phenix: Building New Software for Automated Crystallographic Structure Determination Biological Crystallography Phenix: Building New Software for Automated Crystallographic Structure Determination , 2022 .

[24]  S. Lovett,et al.  Genetic analysis of the recJ gene of Escherichia coli K-12 , 1984, Journal of bacteriology.

[25]  H. Hayashi,et al.  Pre-steady-state kinetics of Escherichia coli aspartate aminotransferase catalyzed reactions and thermodynamic aspects of its substrate specificity. , 1990, Biochemistry.

[26]  S. Lovett,et al.  Reconstitution of Initial Steps of Dsdna Break Repair by the Recf Material Supplemental , 2022 .

[27]  M. Deutscher,et al.  Structural basis for processivity and single-strand specificity of RNase II. , 2006, Molecular cell.

[28]  S. Boulton,et al.  The MRT‐1 nuclease is required for DNA crosslink repair and telomerase activity in vivo in Caenorhabditis elegans , 2009, The EMBO journal.

[29]  M. Schubert,et al.  Structural characterization of the RNase E S1 domain and identification of its oligonucleotide-binding and dimerization interfaces. , 2004, Journal of molecular biology.

[30]  S. Lovett,et al.  Mutational Analysis of the RecJ Exonuclease ofEscherichia coli: Identification of Phosphoesterase Motifs , 1999, Journal of bacteriology.

[31]  B. Matthews.  Solvent content of protein crystals. , 1968, Journal of molecular biology.

[32]  R. Masui,et al.  The crystal structure of exonuclease RecJ bound to Mn2+ ion suggests how its characteristic motifs are involved in exonuclease activity , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[33]  E. Conti,et al.  Structure of the active subunit of the yeast exosome core, Rrp44: diverse modes of substrate recruitment in the RNase II nuclease family. , 2008, Molecular cell.

[34]  B. Matthews,et al.  Type II restriction endonucleases: structural, functional and evolutionary relationships. , 1999, Current opinion in chemical biology.