Structural insight into the substrate specificity of DNA Polymerase μ

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  • A Corrigendum to this article was published on 01 July 2007

Abstract

DNA polymerase μ (Pol μ) is a family X enzyme with unique substrate specificity that contributes to its specialized role in nonhomologous DNA end joining (NHEJ). To investigate Pol μ's unusual substrate specificity, we describe the 2.4 Å crystal structure of the polymerase domain of murine Pol μ bound to gapped DNA with a correct dNTP at the active site. This structure reveals substrate interactions with side chains in Pol μ that differ from other family X members. For example, a single amino acid substitution, H329A, has little effect on template-dependent synthesis by Pol μ from a paired primer terminus, but it reduces both template-independent and template-dependent synthesis during NHEJ of intermediates whose 3′ ends lack complementary template strand nucleotides. These results provide insight into the substrate specificity and differing functions of four closely related mammalian family X DNA polymerases.

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Figure 1: Structure of the murine Pol μ polymerase domain.
Figure 2: Interactions between Pol μ and the gapped primer-template duplex, shown in a stick diagram, colored as in Figure 1.
Figure 3: Comparison of loop conformations between mammalian family X polymerases.
Figure 4: Electrostatic interactions between Pol μ and the bound DNA.
Figure 5: The active site of Pol μ.
Figure 6: Role of Pol μ H329A in catalysis by Pol μ.
Figure 7: Analysis of catalytic activity for Pol μ and TdT.
Figure 8: Role of Pol μ His329 in template-dependent synthesis during nonhomologous end-joining.

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Change history

  • 15 June 2007

    updated crystallization locations

Notes

  1. 1.

    *NOTE: In the version of this article initially published, the crystallization conditions were incorrectly reported. The correct conditions are as follows: 95 mM sodium citrate (pH 5.6), 19% (v/v) isopropanol, 19% (w/v) PEG 4,000 and 5% (v/v) glycerol. The error has been corrected in the HTML and PDF versions of the article. The authors apologize for any inconvenience this may have caused.

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Acknowledgements

We thank T. Hall and S.Nick McElhinny for critical reading and thoughtful comments on the manuscript. This research was funded in part by the Division of Intramural Research of the National Institute of Environmental Health Sciences, US National Institutes of Health, and in part by US National Institutes of Health grant CA097096 to D.A.R. The Advanced Photon Source used for this study was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. W-31-109-Eng-38. We thank Z. Jin for collecting these data at SER-CAT using mail-in crystallography.

Author information

A.F.M., cloning, expression and purification of proteins used for biochemistry and crystallography, crystallization of catalytic domain of murine Pol μ; M.G.-D., expression and purification of proteins to be used for biochemical assays, kinetic analysis of human Pol μ and TdT; K.B., analysis of enzymatic activity for human Pol μ and TdT proteins; B.S.D., analysis of end-joining activity for human Pol μ in NHEJ assays; X.Z., early crystallization trials with human Pol μ; D.A.R., analysis of end-joining activity for human Pol μ and interpretation of data from NHEJ assays; L.C.P., analysis of crystallization data and refinement of Pol μ structural model; T.A.K., experimental design and analysis of biochemical data. All authors contributed to experimental design, interpretation of results and preparation of the manuscript.

Correspondence to Thomas A Kunkel.

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