Artemis Regulates the Mammalian Cell Cycle Via ... · S. cerevisiae mutant: snm1/pso2-sensitivity...
Transcript of Artemis Regulates the Mammalian Cell Cycle Via ... · S. cerevisiae mutant: snm1/pso2-sensitivity...
Artemis Regulates the Mammalian Cell Cycle ViaInteractions with Cullin E3 Ligases
Randy J. Legerski, Ph.D.Department of GeneticsMD Anderson Cancer CenterHouston, TX
S. cerevisiae mutant: snm1/pso2- sensitivity to nitrogen mustard
• Sensitive to bifunctional alkylating agentse.g., nitrogen mustard, cis-platin, psoralen plus UVA
• Not sensitive to IR, UV, or monofunctional alkylating agents
• Proficient in initiation of ICL repair
• Appears to be defective in repair of DSBs created by ICLs
• Has 5’ exonuclease activity on single-stranded DNA
• Mammalian orthologues: SNM1A, SNM1B/Apollo, Artemis
The SNM1 gene of S. cerevisiae
Brief History of ArtemisMoushous et al. Cell 105, 177 (2001)
• Radiosensitive severe combined immunodeficiency syndrome (RS-SCID)
• Defect in V(D)J recombination - lack of T and B cell maturation
• Sensitivity to IR (double-strand breaks)
• Artemis with DNA-PKcs acquires hairpin opening activity during V(D)J recombination
• Phosphorylated by DNA-PKcs in vitroRiballo et al. Mol. Cell 16, 715 (2004)
• Artemis is required in approx. 10% of NHEJ reactions• Artemis is phosphorylated by ATM
Ma et al. Cell 108, 781 (2002)
Beucher et al. EMBO J (2009)• ATM and Artemis promote HR repair of DSBs
All these studies are consistent with the concept of Artemis as a nuclease
Structure of Artemis
• Artemis contains metallo-β-lactamase and β-CASP domains• Artemis is a phospho-protein
Metallo-beta-lactamase
Beta-CASP
516SQ53 4SQ
53 8S Q548SQ553SQ5 62 SQ
6 45SQ
SCD
518SP
385S
692 aa
MBL β-CASP
Artemis and Cellular Stress Responses
DNA damage
IR/DSB Replication block
G2/M checkpointrecovery(cyclin B-Cdk1)
NHEJ HR
ATRATMDNA-PK
S phase checkpoint recovery(cyclin E)
Oxidative stress
DNA-PKcs
p53
Senescenceand apoptosis
Artemis
Zhang et al. Mol. Cell. Biol. (2004)Geng et al. Mol. Cell. Biol. (2007)
Wang et al. JBC (2009)Zhang et al. Oncogene (2009)
IR/DSB Replication block
ATRATM
Artemis
pS534pS538
ArtemispS516
pS645Artemis
pS516
pS645
Cyclin ECyclin B Cdk1
G2/M recovery S phase recovery?
Function of Artemis phosphorylation
Cyclin E is stabilized in response to replication stress
Lu et al. JBC, in press (2009)
p27 Cyc E Cdc25A
Cyclin E degradation is mediated by theSCF-FBW7 E3 ligase
Depletion of Artemis stabilizes cyclin E and delays recovery from the S phase checkpoint
Cel
l cyc
le
phas
e (%
)
1420
66
8
46
46
9
42
49
0
20
40
60
80
100
Con Art1 Art2
G1/S S G2/M
58
24
18
58
29
13
60
32
8
0
20
40
60
80
100
Con Art1 Art2
G1/S S G2/M
55
37
8
58
32
10
55
36
10
0
20
40
6080
100
Con Art1 Art2
G1/S S G2/M
Hrs after releasefrom aphidicolin 3 90
Mutation of S516 and S645 results in prolonged S phase arrest in response to replication stress
Artemis regulates cyclin E stability via ubiquitylation
Artemis interacts with the α and γ isoformsof the F box protein Fbw7
The interaction of Artemis with Fbw7 is regulated by phosphorylation
ATR is required for Artemis-mediated degradation of cyclin E
Conclusions
1. Artemis regulates recovery from the S phase replication checkpoint
2. Artemis interacts with SCFFbw7 to promote degradation of cyclin E
3. Phosphorylation of Artemis at S516 and S645 by ATR in responseto replication stress enhances its association with Fbw7
4. ATR is required for Artemis-mediated degradation of cyclin E
Replication stress ATR ArtemisSCFFbw7
Cyclin E Checkpoint recovery P
Haiyong Wang – graduate student (cyclin E)
Yiyi Yan – graduate student (p27)
Xiaoshan Zhang – instructor
Acknowlegements