PARP1 Val762Ala polymorphism reduces enzymatic activity

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Abstract

Poly(ADP-ribose) polymerase 1 (PARP1) modifies a variety of nuclear proteins by poly(ADP-ribosyl)ation, and plays diverse roles in molecular and cellular processes. A common PARP1 single nucleotide polymorphism (SNP) at codon 762, resulting in the substitution of alanine (Ala) for valine (Val) in the catalytic domain has been implicated in susceptibility to cancer. To characterize the functional effect of this polymorphism on PARP1, we performed in vitro enzymatic analysis on PARP1-Ala762 and PARP1-Val762. We found that PARP1-Ala762 displayed 57.2% of the activity of PARP1-Val762 for auto-poly(ADP-ribosyl)ation and 61.9% of the activity of PARP1-Val762 for trans-poly(ADP-ribosyl)ation of histone H1. The kinetic characterization revealed that the Km of PARP1-Ala762 was increased to a 1.2-fold of the Km of PARP1-Val762 for trans-poly(ADP-ribosyl)ation. Thus, the PARP1 Val762Ala polymorphism reduces the enzymatic activity of PARP1 by increasing Km. This finding suggests that different levels of poly(ADP-ribosyl)ation by PARP1 might aid in understanding the cancer risk of carriers of the PARP1 Val762Ala polymorphism.

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Materials and methods

Expression vector and site-directed mutagenesis. The human PARP1 expression vector pSG9M-PARP1 has been described previously [10]. A point mutation (c. 2446C>T) was introduced into codon 762 of human PARP1 cDNA with a QuikChange® Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA), resulting in an amino acid substitution of valine for alanine. Mutagenesis was performed according to the manufacturer’s recommendation with a sense primer 5′-GTGTGCAGGCCAAGGTGGAAATGCTTGACAACC-3′ and an

Reduced enzymatic activity of PARP1-Ala762 for auto-poly(ADP-ribosyl)ation

To confirm the sequence of human PARP1 cDNA, the expression vector was sequenced. Alignment of the cDNA sequence with the Ensembl transcript (ENST00000272146) of PARP1 revealed three variants. Two are synonymous at codon 192 and 352, and one is non-synonymous at codon 762 resulting in the substitution of alanine for valine. To generate the PARP1–Val762, we introduced a point mutation (c. 2446C>T) at codon 762 of the PARP1 cDNA to substitute valine for alanine in the expression vector.

To compare

Discussion

In this study, we investigated the functional effect of the PARP1 Val762Ala polymorphism on PARP1. We have shown that this polymorphism reduces the enzymatic activity of PARP1 by increasing Km. In our experiments PARP1-Ala762 displayed 57.2% activity of PARP1-Val762 for auto-poly(ADP-ribosyl)ation, and 61.9% activity of PARP1-Val762 for trans-poly(ADP-ribosyl)ation of histone H1, in the presence of activated DNA. For the modification of PARP1 itself and histone H1, the activity decrease of

Acknowledgments

We thank U. Cortes, C. Piccoli, Y. Niu, Y.G. Yang, and C. Cuenin for technical assistance. We are grateful to Prof. Q. Wei for comments and discussions and J. Daniel for editing the manuscript. Further thanks are due to Drs. B. Sylla and E. Van Dyck for reading the manuscript.

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    This work was supported by grants from La Ligue contre le Cancer, Comite du Loire and du Drôme, and Association pour la Researche sur le Cancer, France. X.G. Wang was the recipient of a Special Training Award from the International Agency for Research on Cancer (IARC), France.

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    Present address: Leibniz Institute for Age Research—Fritz Lipmann Institute, Jena, Germany.

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