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Molecular and Cellular Biology, March 2009, p. 1354-1362, Vol. 29, No. 5
0270-7306/09/$08.00+0     doi:10.1128/MCB.01471-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Defective DNA Ligation during Short-Patch Single-Strand Break Repair in Ataxia Oculomotor Apraxia 1 {triangledown}

John J. Reynolds,1,{dagger} Sherif F. El-Khamisy,1,2,{dagger} Sachin Katyal,3 Paula Clements,1 Peter J. McKinnon,3 and Keith W. Caldecott1*

Genome Damage and Stability Centre, University of Sussex, Science Park Road, Falmer, Brighton BN1 9RQ, United Kingdom,1 Biochemistry Department, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt,2 Department of Genetics and Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, Tennessee3

Received 19 September 2008/ Returned for modification 22 October 2008/ Accepted 12 December 2008

Ataxia oculomotor apraxia 1 (AOA1) results from mutations in aprataxin, a component of DNA strand break repair that removes AMP from 5' termini. Despite this, global rates of chromosomal strand break repair are normal in a variety of AOA1 and other aprataxin-defective cells. Here we show that short-patch single-strand break repair (SSBR) in AOA1 cell extracts bypasses the point of aprataxin action at oxidative breaks and stalls at the final step of DNA ligation, resulting in the accumulation of adenylated DNA nicks. Strikingly, this defect results from insufficient levels of nonadenylated DNA ligase, and short-patch SSBR can be restored in AOA1 extracts, independently of aprataxin, by the addition of recombinant DNA ligase. Since adenylated nicks are substrates for long-patch SSBR, we reasoned that this pathway might in part explain the apparent absence of a chromosomal SSBR defect in aprataxin-defective cells. Indeed, whereas chemical inhibition of long-patch repair did not affect SSBR rates in wild-type mouse neural astrocytes, it uncovered a significant defect in Aptx–/– neural astrocytes. These data demonstrate that aprataxin participates in chromosomal SSBR in vivo and suggest that short-patch SSBR arrests in AOA1 because of insufficient nonadenylated DNA ligase.

* Corresponding author. Mailing address: Genome Damage and Stability Centre, University of Sussex, Science Park Road, Falmer, Brighton BN1 9RQ, United Kingdom. Phone: 44 (0) 1273 877519. Fax: 44 (0) 1273 678121. E-mail: k.w.caldecott{at}sussex.ac.uk

{triangledown} Published ahead of print on 22 December 2008.

{dagger} J.J.R. and S.F.E.-K. contributed equally to this work.

Molecular and Cellular Biology, March 2009, p. 1354-1362, Vol. 29, No. 5
0270-7306/09/$08.00+0     doi:10.1128/MCB.01471-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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