Dual Modes of Interaction between XRCC4 and Polynucleotide Kinase/Phosphatase: IMPLICATIONS FOR NONHOMOLOGOUS END JOINING

Rajam S Mani,Yaping Yu,Shujuan Fang,Meiling Lu,Mesfin Fanta,Angela E Zolner,Nasser Tahbaz,Dale A Ramsden,David W Litchfield,Susan P Lees-Miller,Michael Weinfeld

doi:10.1074/jbc.m109.058719

Abstract

XRCC4 plays a crucial role in the nonhomologous end joining (NHEJ) pathway of DNA double-strand break repair acting as a scaffold protein that recruits other NHEJ proteins to double-strand breaks. Phosphorylation of XRCC4 by protein kinase CK2 promotes a high affinity interaction with the forkhead-associated domain of the end-processing enzyme polynucleotide kinase/phosphatase (PNKP). Here we reveal that unphosphorylated XRCC4 also interacts with PNKP through a lower affinity interaction site within the catalytic domain and that this interaction stimulates the turnover of PNKP. Unexpectedly, CK2-phosphorylated XRCC4 inhibited PNKP activity. Moreover, the XRCC4·DNA ligase IV complex also stimulated PNKP enzyme turnover, and this effect was independent of the phosphorylation of XRCC4 at threonine 233. Our results reveal that CK2-mediated phosphorylation of XRCC4 can have different effects on PNKP activity, with implications for the roles of XRCC4 and PNKP in NHEJ.

Highlights

Efficient repair of DNA double-strand breaks (DSBs)3 is critical for the maintenance of genome stability
Previous studies have revealed that polynucleotide kinase/phosphatase (PNKP) interacts with Thr-233-phosphorylated XRCC4 via its N-terminal FHA domain [7], suggesting that PNKP is recruited to DSBs with the XRCC41⁄7DNA ligase IV complex
It is generally considered that interaction between PNKP and these proteins is mediated by the binding of CK2-phosphorylated scaffold protein to the FHA domain of PNKP [7, 31], we have recently shown that XRCC1 can interact with the catalytic domain of PNKP and stimulate PNKP activity [19, 32]

Summary

EXPERIMENTAL PROCEDURES

Expression and Purification of Proteins—Descriptions of the bacterial expression and purification of wild type and mutant human XRCC4 and PNKP are provided in the supplemental materials. PNKP Kinase Activity Assays—PNKP (1.5 ␮g) was premixed with 1.0 or 2.0 ␮g of XRCC4 or pXRCC4 and incubated at 37 °C for 5 min and added to a reaction mixture (20 ␮l of total volume) containing kinase buffer (80 mM succinic acid, pH 5.5, 10 mM MgCl2, and 1 mM DTT), 2 ␮M 45-bp duplex DNA substrate, and 3.3 pmol of [␥-32P]ATP (PerkinElmer Life Sciences) and incubated for 20 min at 37 °C. For immunoprecipitation of transfected proteins, 72 h after transfection, the cells were lysed by incubation on ice for 30 min in HEPES/Nonidet P-40 lysis buffer (40 mM HEPES, pH7.5, 1 mM EDTA, 50 mM NaCl, 0.1% (v/v) Nonidet P-40) containing 0.2 ␮M phenylmethylsulfonyl fluoride, 0.2 ␮g/ml leupeptin, 0.2 ␮g/ml aprotinin, 0.2 ␮g/ml pepstatin, and 1 ␮M microcystin-LR, followed by sonication. The beads were washed five times with 1 ml of low salt/low detergent NETN buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 50 mM NaCl, 0.1% (v/v) Nonidet P-40, 0.2 mM phenylmethylsulfonyl fluoride) and analyzed by SDS-PAGE and immunoblot as indicated

RESULTS

11 Ϯ 1 13 Ϯ 2 15 Ϯ 2 14 Ϯ 2

DISCUSSION