Encounter and extrusion of an intrahelical lesion by a DNA repair enzyme

Abstract

How living systems detect the presence of genotoxic damage embedded in a million-fold excess of undamaged DNA is an unresolved question in biology. Here we have captured and structurally elucidated a base-excision DNA repair enzyme, MutM, at the stage of initial encounter with a damaged nucleobase, 8-oxoguanine (oxoG), nested within a DNA duplex. Three structures of intrahelical oxoG-encounter complexes are compared with sequence-matched structures containing a normal G base in place of an oxoG lesion. Although the protein–DNA interfaces in the matched complexes differ by only two atoms—those that distinguish oxoG from G—their pronounced structural differences indicate that MutM can detect a lesion in DNA even at the earliest stages of encounter. All-atom computer simulations show the pathway by which encounter of the enzyme with the lesion causes extrusion from the DNA duplex, and they elucidate the critical free energy difference between oxoG and G along the extrusion pathway.

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Figure 1: Generation and recognition of 8-oxoguanine.
Figure 2: Helix-penetration by MutM residues.
Figure 3: OxoG-dependent local DNA structure alterations at the site of the target base in EC3 and EC5.
Figure 4: Free energy profiles of nucleobase extrusion imply active participation of MutM.
Figure 5: R112-catalysed oxoG extrusion.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the reported crystal structures have been deposited with the Protein Data Bank under accession codes 3GPY (LRC3), 3GO8 (EC3), 3GP1 (EC3V222P), 3GPP (EC3T224P), 3GPU (EC4), 3GPX (IC4), 3GQ4 (LRC5), 3GQ3(EC5) and 3GQ5 (IC5).

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Acknowledgements

This work was supported by grants from the NIH: GM044853, GM047467, CA100742 (G.L.V.) and GM030804 (M.K.). Y.Q. is supported by a predoctoral fellowship from the National Science Foundation; M.C.S. by a predoctoral fellowship from the Howard Hughes Medical Institute; K.N. by a postdoctoral fellowship from National Cancer Center. We thank the staff of the NSLS, APS and CHESS synchrotron facilities. These experiments made use of the computing facilities at NERSC and Harvard FAS. We are grateful to J. Jin for experimental assistance and J. Pu, V. Ovchinnikov and other members of the Karplus and Verdine groups for helpful advice.

Author Contributions Y.Q., M.C.S., K.N. and A.B. contributed equally to the study. A.B., S.J. and M.C.S. cloned the constructs. A.B., M.C.S. and S.J. performed the biochemical and FP assays. A.B., S.J., M.C.S. and Y.Q. purified, crystallized and collected X-ray diffraction data and solved structures (A.B., S.J.: EC4, EC5; M.C.S.: EC3, EC3V222P, EC3T224P, IC4, LRC5; Y.Q.: LRC3, EC3T224P, EC5, IC5, LRC5). A.B. and G.L.V. designed the trapping strategy and crystallographic studies. K.N. and M.K. designed the computational studies, which K.N. then performed. A.B., M.C.S., Y.Q., K.N., G.L.V. and M.K. analysed data and wrote the paper. G.L.V. and M.K. directed the research. All authors discussed the results and commented on the manuscript.

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Correspondence to Martin Karplus or Gregory L. Verdine.

Supplementary information

Supplementary Information

This file contains Supplementary Notes, Supplementary References, Supplementary Figures 1-21 with Legends and Supplementary Tables S1- S3. (PDF 9032 kb)

Supplementary Movie 1

This movie file shows the MutM-assisted base extrusion pathway calculated using the EC4 crystal structure. The target base is oxoG. (MOV 6863 kb)

Supplementary Movie 2

This movie file shows the MutM-assisted base extrusion pathway calculated using the IC4 crystal structure. The target base is G. (MOV 6548 kb)

Supplementary Movie 3

This movie file shows the MutM-assisted base extrusion pathway calculated using the EC5 crystal structure. The target base is oxoG. (MOV 6869 kb)

Supplementary Movie 4

This movie file shows the MutM-assisted base extrusion pathway calculated using the IC5 crystal structure. The target base is G. (MOV 6616 kb)

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Qi, Y., Spong, M., Nam, K. et al. Encounter and extrusion of an intrahelical lesion by a DNA repair enzyme. Nature 462, 762–766 (2009). https://doi.org/10.1038/nature08561

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