Chk1 both arrests replication forks and enhances repair of DNA damage by 
phosphorylating downstream effectors. Although there has been a concerted effort 
to identify effectors of Chk1 activity, underlying mechanisms of effector action 
are still being identified. Metnase (also called SETMAR) is a SET and 
transposase domain protein that promotes both DNA double-strand break (DSB) 
repair and restart of stalled replication forks. In this study, we show that 
Metnase is phosphorylated only on Ser495 (S495) in vivo in response to DNA 
damage by ionizing radiation. Chk1 is the major mediator of this phosphorylation 
event. We had previously shown that wild-type (wt) Metnase associates with 
chromatin near DSBs and methylates histone H3 Lys36. Here we show that a 
Ser495Ala (S495A) Metnase mutant, which is not phosphorylated by Chk1, is 
defective in DSB-induced chromatin association. The S495A mutant also fails to 
enhance repair of an induced DSB when compared with wt Metnase. Interestingly, 
the S495A mutant demonstrated increased restart of stalled replication forks 
compared with wt Metnase. Thus, phosphorylation of Metnase S495 differentiates 
between these two functions, enhancing DSB repair and repressing replication 
fork restart. In summary, these data lend insight into the mechanism by which 
Chk1 enhances repair of DNA damage while at the same time repressing stalled 
replication fork restart.