α-N-Methylation of Damaged DNA-binding Protein 2 (DDB2) and Its Function in Nucleotide Excision Repair

Qian Cai,Lijuan Fu,Zi Wang,Nanqin Gan,Xiaoxia Dai,Yinsheng Wang

doi:10.1074/jbc.m114.558510

Abstract

DDB2 exhibits a high affinity toward UV-damaged DNA, and it is involved in the initial steps of global genome nucleotide excision repair. Mutations in the DDB2 gene cause the genetic complementation group E of xeroderma pigmentosum, an autosomal recessive disease manifested clinically by hypersensitivity to sunlight exposure and an increased predisposition to skin cancer. Here we found that, in human cells, the initiating methionine residue in DDB2 was removed and that the N-terminal alanine could be methylated on its α-amino group in human cells, with trimethylation being the major form. We also demonstrated that the α-N-methylation of DDB2 is catalyzed by the N-terminal RCC1 methyltransferase. In addition, a methylation-defective mutant of DDB2 displayed diminished nuclear localization and was recruited at a reduced efficiency to UV-induced cyclobutane pyrimidine dimer foci. Moreover, loss of this methylation conferred compromised ATM (ataxia telangiectasia mutated) activation, decreased efficiency in cyclobutane pyrimidine dimer repair, and elevated sensitivity of cells toward UV light exposure. Our study provides new knowledge about the posttranslational regulation of DDB2 and expands the biological functions of protein α-N-methylation to DNA repair.

Highlights

Because its N terminus adopts an APK motif, DDB2 might be ␣-N-methylated
DDB2 could be ␣-N-methylated by NRMT, and this methylation facilitated the recruitment of DDB2 to DNA damage foci
␣-N-Methylation of DDB2 Facilitates cyclobutane pyrimidine dimer (CPD) Repair in GM01389 Cells—Seeing that the methylation-defective mutant of DDB2 exhibited diminished nuclear localization and reduced recruitment to CPD foci, we examined whether the ␣-N-methylation of DDB2 plays a role in CPD repair using a flow cytometry-based method [30]

Summary

Background

Because its N terminus adopts an APK motif, DDB2 might be ␣-N-methylated. Results: We examined the nature of DDB2 ␣-N-methylation, the enzyme involved in this methylation and its function in DNA repair. A methylation-defective mutant of DDB2 displayed diminished nuclear localization and was recruited at a reduced efficiency to UVinduced cyclobutane pyrimidine dimer foci Loss of this methylation conferred compromised ATM (ataxia telangiectasia mutated) activation, decreased efficiency in cyclobutane pyrimidine dimer repair, and elevated sensitivity of cells toward UV light exposure. Our study provides new knowledge about the posttranslational regulation of DDB2 and expands the biological functions of protein ␣-N-methylation to DNA repair. We demonstrated that NRMT could catalyze the ␣-N-methylation of DDB2 in vitro and in human cells and that this methylation promoted the nuclear localization of DDB2, facilitated the recruitment of DDB2 to CPD foci, augmented CPD repair efficiency, enabled ATM activation, and conferred resistance of human cells toward UV damage. Results from our study expand the function of protein ␣-N-methylation to DNA repair

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION