Chk1 phosphorylation of Metnase enhances DNA repair but inhibits replication fork restart.
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.
Department of Medicine, University of Florida and Shands Health Care System, Gainesville, FL 32610, USA. robert.hromas@medicine.ufl.edu
R
R
Hromas
E A
EA
Williamson
S
S
Fnu
Y-J
YJ
Lee
S-J
SJ
Park
B D
BD
Beck
J-S
JS
You
A
A
Leitao
A
A
Laitao
J A
JA
Nickoloff
S-H
SH
Lee
D045744:Cell Line, Tumor; D000071877:Checkpoint Kinase 1; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D057809:HEK293 Cells; D011495:Histone-Lysine N-Methyltransferase / Q000235:genetics / Q000378:metabolism*; D006657:Histones; D006801:Humans; D008745:Methylation; D009154:Mutation; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*
D006657:Histones; D011495:Histone-Lysine N-Methyltransferase; C510450:SETMAR protein, human; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1