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Kim, HS, Chen, Q, Kim, SK, Nickoloff, JA, Hromas, R, Georgiadis, MM and Lee, SH (2014) The DDN catalytic motif is required for Metnase functions in non-homologous end joining (NHEJ) repair and replication restart. J. Biol. Chem. 289:10930-8

Metnase (or SETMAR) arose from a chimeric fusion of the Hsmar1 transposase downstream of a protein methylase in anthropoid primates. Although the Metnase transposase domain has been largely conserved, its catalytic motif (DDN) differs from the DDD motif of related transposases, which may be important for its role as a DNA repair factor and its enzymatic activities. Here, we show that substitution of DDN(610) with either DDD(610) or DDE(610) significantly reduced in vivo functions of Metnase in NHEJ repair and accelerated restart of replication forks. We next tested whether the DDD or DDE mutants cleave single-strand extensions and flaps in partial duplex DNA and pseudo-Tyr structures that mimic stalled replication forks. Neither substrate is cleaved by the DDD or DDE mutant, under the conditions where wild-type Metnase effectively cleaves ssDNA overhangs. We then characterized the ssDNA-binding activity of the Metnase transposase domain and found that the catalytic domain binds ssDNA but not dsDNA, whereas dsDNA binding activity resides in the helix-turn-helix DNA binding domain. Substitution of Asn-610 with either Asp or Glu within the transposase domain significantly reduces ssDNA binding activity. Collectively, our results suggest that a single mutation DDN(610) → DDD(610), which restores the ancestral catalytic site, results in loss of function in Metnase.

PubMed PMC4036204 Online version:10.1074/jbc.M113.533216

Amino Acid Motifs; Asparagine/chemistry; Base Sequence; Catalytic Domain; Cell Nucleus/metabolism; DNA End-Joining Repair; DNA Replication; DNA, Single-Stranded/chemistry; DNA-Binding Proteins/metabolism; HEK293 Cells; Histone-Lysine N-Methyltransferase/chemistry; Histones/chemistry; Humans; Molecular Sequence Data; Protein Binding; RNA Interference; Transposases/metabolism