Limited terminal transferase in human DNA polymerase μ defines the required balance between accuracy and efficiency in NHEJ

DNA polymerase mu (Polμ) is a family X member implicated in DNA repair, with template-directed and terminal transferase (template-independent) activities. It has been proposed that the terminal transferase activity of Polμ can be specifically required during non-homologous end joining (NHEJ) to create or increase complementarity of DNA ends. By site-directed mutagenesis in human Polμ, we have identified a specific DNA ligand residue (Arg387) that is responsible for its limited terminal transferase activity compared to that of human TdT, its closest homologue (42% amino acid identity). Polμ mutant R387K (mimicking TdT) displayed a large increase in terminal transferase activity, but a weakened interaction with ssDNA. That paradox can be explained by the regulatory role of Arg387 in the translocation of the primer from a non-productive E:DNA complex to a productive E:DNA:dNTP complex in the absence of a templating base, which is probably the rate limiting step during template-independent synthesis. Further, we show that the Polμ switch from terminal transferase to templated insertions in NHEJ reactions is triggered by recognition of a 5′-P at a second DNA end, whose 3′-protrusion could provide a templating base to facilitate such a special “pre-catalytic translocation step.” These studies shed light on the mechanism by which a rate-limited terminal transferase activity in Polμ could regulate the balance between accuracy and necessary efficiency, providing some variability during NHEJ.

[1]  S. Tonegawa,et al.  Somatic generation of antibody diversity. , 1976, Nature.

[2]  C. M. Joyce,et al.  Novel blunt-end addition reactions catalyzed by DNA polymerase I of Escherichia coli. , 1987, Journal of molecular biology.

[3]  J. Clark,et al.  Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. , 1988, Nucleic acids research.

[4]  T. Lohman,et al.  A double-filter method for nitrocellulose-filter binding: application to protein-nucleic acid interactions. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[5]  J. Mornon,et al.  From BRCA1 to RAP1: a widespread BRCT module closely associated with DNA repair , 1997, FEBS letters.

[6]  Peer Bork,et al.  A superfamily of conserved domains in DNA damage‐ responsive cell cycle checkpoint proteins , 1997, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[7]  K. Mahajan,et al.  Association of terminal deoxynucleotidyl transferase with Ku. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[8]  T. Kirchhoff,et al.  DNA polymerase lambda (Pol lambda), a novel eukaryotic DNA polymerase with a potential role in meiosis. , 2000, Journal of molecular biology.

[9]  T. Kirchhoff,et al.  DNA polymerase lambda (Pol λ), a novel eukaryotic DNA polymerase with a potential role in meiosis. , 2000 .

[10]  T. Kirchhoff,et al.  DNA polymerase mu (Pol mu), homologous to TdT, could act as a DNA mutator in eukaryotic cells. , 2000, The EMBO journal.

[11]  M. García-Díaz,et al.  DNA polymerase mu, a candidate hypermutase? , 2001, Philosophical transactions of the Royal Society of London. Series B, Biological sciences.

[12]  S. Hughes,et al.  Nontemplated nucleotide addition by HIV-1 reverse transcriptase. , 2002, Biochemistry.

[13]  C. Papanicolaou,et al.  Crystal structures of a template‐independent DNA polymerase: murine terminal deoxynucleotidyltransferase , 2002, The EMBO journal.

[14]  D. Ramsden,et al.  Association of DNA polymerase mu (pol mu) with Ku and ligase IV: role for pol mu in end-joining double-strand break repair. , 2002, Molecular and cellular biology.

[15]  D. Ramsden,et al.  Association of DNA Polymerase μ (pol μ) with Ku and Ligase IV: Role for pol μ in End-Joining Double-Strand Break Repair , 2002, Molecular and Cellular Biology.

[16]  M. Piris,et al.  Overexpression of human DNA polymerase mu (Pol mu) in a Burkitt's lymphoma cell line affects the somatic hypermutation rate. , 2004, Nucleic acids research.

[17]  T. Kunkel,et al.  Functions of DNA polymerases. , 2004, Advances in protein chemistry.

[18]  Yunmei Ma,et al.  A biochemically defined system for mammalian nonhomologous DNA end joining. , 2004, Molecular cell.

[19]  T. Kunkel,et al.  A gradient of template dependence defines distinct biological roles for family X polymerases in nonhomologous end joining. , 2005, Molecular cell.

[20]  D. Ramsden,et al.  A specific loop in human DNA polymerase mu allows switching between creative and DNA-instructed synthesis , 2006, Nucleic acids research.

[21]  E. Friedberg,et al.  DNA Repair and Mutagenesis , 2006 .

[22]  B. Bertocci,et al.  Nonoverlapping functions of DNA polymerases mu, lambda, and terminal deoxynucleotidyltransferase during immunoglobulin V(D)J recombination in vivo. , 2006, Immunity.

[23]  M. Lieber,et al.  XRCC4:DNA ligase IV can ligate incompatible DNA ends and can ligate across gaps , 2007, The EMBO journal.

[24]  T. Kunkel,et al.  Structural insight into the substrate specificity of DNA Polymerase mu. , 2007, Nature structural & molecular biology.

[25]  Samuel H. Wilson,et al.  The X family portrait: structural insights into biological functions of X family polymerases. , 2007, DNA repair.

[26]  T. Kunkel,et al.  Structural insight into the substrate specificity of DNA Polymerase μ , 2007, Nature Structural &Molecular Biology.

[27]  D. Ramsden,et al.  End-bridging is required for pol μ to efficiently promote repair of noncomplementary ends by nonhomologous end joining , 2008, Nucleic acids research.

[28]  L. Blanco,et al.  A Role for DNA Polymerase μ in the Emerging DJH Rearrangements of the Postgastrulation Mouse Embryo , 2008, Molecular and Cellular Biology.