Solution structure of the single-strand break repair protein XRCC1 N-terminal domain

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Abstract

XRCC1 functions in the repair of single-strand DNA breaks in mammalian cells and forms a repair complex with β-Pol, ligase III and PARP. Here we describe the NMR solution structure of the XRCC1 N-terminal domain (XRCC1 NTD). The structural core is a β-sandwich with β-strands connected by loops, three helices and two short two-stranded β-sheets at each connection side. We show, for the first time, that the XRCC1 NTD specifically binds single-strand break DNA (gapped and nicked). We also show that the XRCC1 NTD binds a gapped DNA–β-Pol complex. The DNA binding and β-Pol binding surfaces were mapped by NMR and found to be well suited for interaction with single-strand gap DNA containing a 90° bend, and for simultaneously making contacts with the palm-thumb of β-Pol in a ternary complex. The findings suggest a mechanism for preferential binding of the XRCC1 NTD to flexible single-strand break DNA.

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Figure 1
Figure 2: The NMR solution structure of the XRCC1 NTD.
Figure 3: Topology and sequences of XRCC1 NTD.
Figure 4: Electrostatic surface potential shown with positive charge colored blue and negative charge colored red.
Figure 5: Electrophoretic gel mobility shift assay of XRCC1 NTD binding to single-strand break DNA and to a single-nucleotide gap–β-Pol complex.
Figure 6: Mapping the DNA and β-Pol interaction surfaces on the XRCC1 NTD by NMR chemical shift changes.
Figure 7: XRCC1 NTD binding to a 26-mer duplex DNA containing a single-nucleotide gap and chemical shift mapping of the 1H and 15N amide chemical shift changes.
Figure 8: The postulated interaction mode of the XRCC1 NTD with the single-nucleotide gap DNA–β-Pol complex and single-strand break DNA.
Figure 9: Ribbon overlay of the β-strands in the XRCC1 NTD (violet) and the galactose binding domain (yellow) from sialidase (accession code 1EUU).

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Acknowledgements

We thank A. Robertson and S. Wilson for providing the overexpression clone of the palm-thumb domain of β-Pol. We thank M. Santos for synthesis of the oligonucleotides used for NMR studies. This research was supported by an NIH grant to G.P.M. and by NIH postdoctoral fellowship grants to M.W.M. and to B.P.

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Correspondence to Gregory P. Mullen.

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