The DDN Catalytic Motif is required for Metnase Functions in NHEJ Repair and Replication Restart [Enzymology]

February 25th, 2014 by Kim, H.-S., Chen, Q., Kim, S.-K., Nickoloff, J. A., Hromas, R., Georgiadis, M. M., Lee, S.-H.

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 DDN610 with either DDD610 or DDE610 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-Y 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, while dsDNA binding activity resides in the helix-turn-helix DNA binding domain. Substitution of N610 with either D or E within the transposase domain significantly reduces ssDNA binding activity. Collectively, our results suggest that a single mutation DDN610 → DDD610, which restores the ancestral catalytic site, results in loss of function in Metnase.