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