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Nrm1 and DNA Stress

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Injuries to DNA that introduce deviation from itsnormal intact structure

If left unrepaired ,results in a mutation or a block ofDNA replication

Caused by physical and chemical reagents

Includes modification of bases,loss of base,singlestrand breaks,crosslinking etc

If not repaired leads to Apoptosis

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Rad 3

ProteinKinase

Cds1 & Chk1Checkpointeffector

protein kinase

MBF

Transcription

Factor

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Mlu1 cell cycle box(MCB) Binding Factor

Sequence specific DNAbinding Proteins

Cdc10

+

Res1

Res2

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Mass Spectroscopy-basedMultidimensional Protein Interaction

Technology(MuDPIT) analysis of affinity-purified Res2 and Nrm1

Results

Nrm1 is a component of MBF

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Nrm1 does not interact with Res1 if Res2 isdeleted and vice-versa

Binds to cdc10 component in either case cdc10-C4 mutant abrogates the binding

Interpretation

Nrm1 binds to cdc10 component of MBF

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Binds to the promoter of G1-S specificgenes required for DNA repair

Transcription of these genes will

produce proteins required forcorrection of DNA damage

Activation of DNA checkpoints should

activate these genes Main checkpoint being DNA replication

checkpoint

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cdc10-C4 will not allow Nrm1 to bindto MBF

will allow MBF to bind to thepromoter

Observation Constitutively expressed MBF target

genes throughout the cell cycle

Interpretation Nrm1 required for repression of MBF

target genes

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Remove Res2

Observation Nrm1 binds to MBF

Binding did not repress the transcription

Interpretation Binding of Nrm1 to DNA requires intact

MBF

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1.Treat the cells with Hydroxyurea (HU)

Observation

Nrm1 accumulated in the cells as a

slow migrating form that did not bindto the promoters

2. Release the cells from HU

Observation Nrm1 shifted to faster migrating form

repression of transcription

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Isolate Nrm1 from HU-arrested cells

Phosphatase treatment

Observation Slow migrating form got converted to

fast moving form

Interpretation Phosphorylation of Nrm1 is

responsible for its inactivation

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Treat the cells with agents that blockreplication fork progression

Observation

Nrm1 phosphorylation andTranscriptional induction

Interpretation Nrm1 gets phosphorylated during

DNA Replication Stress

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Mutate the checkpoint protein kinases1.Take cds1 mutant Treat with HU

Observation

Nrm1 phosphorylation reduced Transcription decreased as compared to wild

type

But not substantially decreased as expectedPossible reason

Activation of chk12. Inactivate chk1 also- increased repression

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Isolate and purify Cds11.Treat Nrm1 with Cds1 using labelled ATP Observation Incorporation of labelled phosphate detected in

Nrm1 Quantitative increase in slower mobility species of

Nrm1 detected2. Incubate Nrm1 with catalytically inactive Cds1 Observation No Phosphorylation Interpretation Nrm1 is a direct target for Cds1

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G1-S transcription factors and their regulators areputative targets of cell cycle checkpoints thatregulate genomic stability

Expected to play a central role in the avoidance ofDNA damage and chromosomal aberrations,phenomena that directly contribute totumorigenesis

Understanding the mechanisms governingregulation of G1-S gene expression in response togenotoxic stress may provide new insights into the

genesis and treatment of human cancer

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Rhind N, Russell P (2000) Chk1 and Cds1: linchpins of the DNA damageand replication checkpoint pathways.J Cell Sci 113:38893896

Chu Z, Li J, Eshaghi M, Peng X, Karuturi RK, Liu J (2007) Modulation of cellcycle-specific gene expressions at the onset of S phase arrest contributesto the robust DNA replication checkpoint response in fission yeast. Mol BiolCell 18:17561767

Boddy MN, Furnari B, Mondesert O, Russell P (1998) Replicationcheckpoint enforced by kinases Cds1 and Chk1. Science 280:909912

Zeng Y, Forbes KC, Wu Z, Moreno S, Piwnica-Worms H, Enoch T (1998)Replication checkpoint requires phosphorylation of the phosphataseCdc25 by Cds1 or Chk1 Nature 395:507510

R. A. M. de Bruin, T. I. Kalashnikova, A. Aslanian, J. Wohlschlegel, C.Chahwan, J. R. Yates III, P. Russell,and C. Wittenberg(2008) DNA

replication checkpoint promotes G1-S transcription by inactivating theMBF repressor Nrm1 PNAS 105: 11230-11235