Requirements for 5′dRP/AP lyase activity in Ku

Natasha T Strande; Juan Carvajal-Garcia; Ryan A Hallett; Crystal A Waters; Steven A Roberts; Christina Strom; Brian Kuhlman; Dale A Ramsden

doi:10.1093/nar/gku796

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Requirements for 5′dRP/AP lyase activity in Ku

Journal article

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Requirements for 5′dRP/AP lyase activity in Ku

Natasha T Strande, Juan Carvajal-Garcia, Ryan A Hallett, Crystal A Waters, Steven A Roberts, Christina Strom, Brian Kuhlman and Dale A Ramsden

Nucleic acids research, Vol.42(17), pp.11136-11143

09/29/2014

DOI: https://doi.org/10.1093/nar/gku796

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https://hdl.handle.net/2376/100710

PMCID: PMC4176175

PMID: 25200085

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https://doi.org/10.1093/nar/gku796View

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Abstract

Nucleic Acid Enzymes

The non-homologous end joining (NHEJ) pathway is used in diverse species to repair chromosome breaks, and is defined in part by a requirement for Ku. We previously demonstrated mammalian Ku has intrinsic 5′ deoxyribosephosphate (5′dRP) and apurinic/apyrimidinic (AP) lyase activity, and showed this activity is important for excising abasic site damage from ends. Here we employ systematic mutagenesis to clarify the protein requirements for this activity. We identify lysine 31 in the 70 kD subunit (Ku70 K31) as the primary candidate nucleophile required for catalysis, but additional mutation of Ku70 K160 and six other lysines within Ku80 were required to eliminate all activity. Ku from Saccharomyces cerevisiae also possesses 5′dRP/AP lyase activity, and robust activity was also reliant on lysines in Ku70 analogous to K31 and K160. By comparison, these lysines are not conserved in Xenopus laevis Ku, and Ku from this species has negligible activity. A role for residues flanking Ku70 K31 in expanding the range of abasic site contexts that can be used as substrate was also identified. Our results suggest an active site well located to provide the substrate specificity required for its biological role.

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Title: Requirements for 5′dRP/AP lyase activity in Ku
Creators: Natasha T Strande - Lineberger Comprehensive Cancer Center, Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27514, USA
Juan Carvajal-Garcia - Lineberger Comprehensive Cancer Center, Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27514, USA
Ryan A Hallett - Lineberger Comprehensive Cancer Center, Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27514, USA
Crystal A Waters - Lineberger Comprehensive Cancer Center, Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27514, USA
Steven A Roberts - Lineberger Comprehensive Cancer Center, Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27514, USA
Christina Strom - Lineberger Comprehensive Cancer Center, Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27514, USA
Brian Kuhlman - Lineberger Comprehensive Cancer Center, Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27514, USA
Dale A Ramsden - Lineberger Comprehensive Cancer Center, Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27514, USA
Publication Details: Nucleic acids research, Vol.42(17), pp.11136-11143
Academic Unit: Molecular Biosciences, School of
Publisher: Oxford University Press
Identifiers: 99900546560001842
Language: English
Resource Type: Journal article