The human set and transposase domain protein Metnase interacts with DNA Ligase IV and enhances the efficiency and accuracy of non-homologous end-joining

Abstract

Transposase domain proteins mediate DNA movement from one location in the genome to another in lower organisms. However, in human cells such DNA mobility would be deleterious, and therefore the vast majority of transposase-related sequences in humans are pseudogenes. We recently isolated and characterized a SET and transposase domain protein termed Metnase that promotes DNA double-strand break (DSB) repair by non-homologous end-joining (NHEJ). Both the SET and transposase domain were required for its NHEJ activity. In this study we found that Metnase interacts with DNA Ligase IV, an important component of the classical NHEJ pathway. We investigated whether Metnase had structural requirements of the free DNA ends for NHEJ repair, and found that Metnase assists in joining all types of free DNA ends equally well. Metnase also prevents long deletions from processing of the free DNA ends, and improves the accuracy of NHEJ. Metnase levels correlate with the speed of disappearance of gamma-H2Ax sites after ionizing radiation. However, Metnase has little effect on homologous recombination repair of a single DSB. Altogether, these results fit a model where Metnase plays a role in the fate of free DNA ends during NHEJ repair of DSBs.

Fig. 1. Metnase interacts with DNA Ligase IV and XRCC4

Extracts from HEK-293 cells expressing V5-tagged Metnase and Flag-tagged DNA Ligase IV were immunoprecipitated with Protein-G beads and anti-Flag (panel A) or anti-V5 antibodies (panel B); mock-IP controls without antibodies are indicated by “Beads.” Immunoprecipitates were separated by PAGE and Western blots were probed with anti-V5 or anti-Flag antibodies. IR increases the interaction between Metnase and DNA Ligase IV when DNA is present. (C, D) Immunoprecipitation as above except samples were treated with DNase I. In panel D the anti-V5 immunoprecipitate was also probed with anti-XRCC4 antibodies. (E) Extracts from HEK-293 cells expressing wither V5-tagged Metnase or Flag-tagged DNA Ligase IV were immunoprecipitated with Protein-G beads and anti-V5 or anti-Flag antibodies. Immunoprecipitates were probed with anti-DNA Ligase IV or anti-Metnase.

Fig. 2. Cell lines that over- and under-express Metnase

(A) RT-PCR using Metnase-specific primers with 18S rRNA as loading control. (B) Western immunoblots with an anti-Metnase antibody and anti-β-actin as loading control. Vector: controls; Metnase: Metnase over-expressing cells; si-Metnase: under-expressing cells.

Fig. 3. Metnase promotes plasmid NHEJ with 5’ and 3’ overhangs and blunt ends in a One Step assay

(A) Metnase over-expression increases precise and imprecise NHEJ. (B) Metnase under-expression decreases precise and imprecise NHEJ. Values are averages (±SEM) of 3 distinct determinations. **P < 0.01.

Fig. 4. Metnase prevents large deletions during NHEJ

(A) Metnase over-expression decreases the frequency of deletions >90 bp in length during NHEJ of 5’ overhangs and blunt ends; *P = 0.027; **P = 0.0066. (B) Metnase under-expression increases the frequency of large deletions during NHEJ of 5’ overhangs. Values as in Fig. 3; * indicates P = 0.021. The number of repair products tested is shown below each graph.

Fig. 5. Over-expression of Metnase enhances NHEJ accuracy and efficiency in a Two Step coupled NHEJ/integration assay

(A) Over-expression of Metnase increases the number of hygromycin-resistant colonies; averages ± SEM for 9 determinations. (B, C) Metnase over-expression decreases the frequency of large deletions and increases precise repair; 40–104 products tested per condition, **P < 0.0001 by Fisher exact tests.

Fig. 6. Metnase does not suppress HR

HT256 co-transfected with control vector or pcDNA-Metnase, and I-SceI sense or control vector and G418-resistant colonies were scored. Values as in Fig. 5A.

Fig. 7. Metnase promotes resolution of γ-H2Ax foci after IR

(A) Representative images of γ-H2Ax foci (green) with DAPI (blue) in control (unirradiated) cells, and 0.25 or 6 hr after IR-treated cells with normal and over-expressed Metnase. (B) As in panel A except control and Metnase under-expression are shown. (C) The percentages γ-H2Ax-positive cells were counted with no IR (averaging ~5% regardless of Metnase expression level), and at 0.25 and 6 hr after 2 Gy IR. At 0.25 hr, >75% of cells had γ-H2Ax foci and this number declined after 6 hr. Plotted are the ratios of γ-H2Ax-positive cells at 6 hr divided by 0.25 hr, with control values normalized to 100%.