The SET domain protein Metnase mediates foreign DNA integration and links integration to nonhomologous end-joining repair

Abstract

The molecular mechanism by which foreign DNA integrates into the human genome is poorly understood yet critical to many disease processes, including retroviral infection and carcinogenesis, and to gene therapy. We hypothesized that the mechanism of genomic integration may be similar to transposition in lower organisms. We identified a protein, termed Metnase, that has a SET domain and a transposase͞nuclease domain. Metnase methylates histone H3 lysines 4 and 36, which are associated with open chromatin. Metnase increases resistance to ionizing radiation and increases nonhomologous end-joining repair of DNA doublestrand breaks. Most significantly, Metnase promotes integration of exogenous DNA into the genomes of host cells. Therefore, Metnase is a nonhomologous end-joining repair protein that regulates genomic integration of exogenous DNA and establishes a relationship among histone modification, DNA repair, and integration. The data suggest a model wherein Metnase promotes integration of exogenous DNA by opening chromatin and facilitating joining of DNA ends. This study demonstrates that eukaryotic transposase domains can have important cell functions beyond transposition of genetic elements.DNA repair ͉ histone methylation

Expression levels of the human DNA repair protein metnase influence lentiviral genomic integration

“…Metnase contains two functional domains: a SET domain and a Transposase/Integrase domain, which contains a DDE motif that is common to the transposase and retroviral integrase family. We found that Metnase under-expression resulted both in decreased DNA DSB repair by NHEJ and decreased integration of foreign DNA [1]. Genomic integration of lentiviruses such as HIV has three distinct steps.…”

“…HEK293 cells were transfected with pcDNA3.1-Metnase or pcDNA3.1 (vector control, Invitrogen, Carlsbad, CA) and selected with G418 for stable over-expression of Metnase as described previously (1). For Metnase under-expression HEK293 cells were transfected with pRNA-U6/hygro-siMET or pRNA-U6/hygro (vector control expressing a scrambled siRNA; Genscript, Piscataway, NJ), selected with hygromycin and individual colonies were screened for Metnase under-expression by reverse transcription-PCR (RT-PCR) [1].…”

“…For Metnase under-expression HEK293 cells were transfected with pRNA-U6/hygro-siMET or pRNA-U6/hygro (vector control expressing a scrambled siRNA; Genscript, Piscataway, NJ), selected with hygromycin and individual colonies were screened for Metnase under-expression by reverse transcription-PCR (RT-PCR) [1]. HEK293 cells were also stably transfected with pCMV-FLAG (vector control, Sigma-Aldrich, Milwaukee, WI) and pCMV-FLAG constructs of wild type Metnase or the Metnase mutant D483A, which changes the first D in the conserved DDE motif required for transposase and HIV integrase function [1]. Expression for all lines was monitored before each experiment by Western immunoblotting [1].…”

“…In a previous study we identified a novel protein termed Metnase, which promotes double strand break (DSB) repair by non-homologous end-joining (NHEJ) and is important for the integration of foreign DNA into the host cell genome [1]. Metnase contains two functional domains: a SET domain and a Transposase/Integrase domain, which contains a DDE motif that is common to the transposase and retroviral integrase family.…”

“…The completion of the integration process by repairing the gapped DNA intermediates may involve a number of DNA repair proteins, including the NHEJ repair pathway [2][3][4], Poly (ADP-ribose)-polymerase (PARP) [5,6], ATM [7], ATR [8] and RAD18 proteins [9]. Our previous study demonstrated that Metnase was important for integration of free fragments of foreign DNA [1]. Given that Metnase contained a domain similar to transposase and integrase family members, we therefore examined whether Metnase played a role in lentiviral genomic integration.…”