Cellular Responses to DNA Damage

Kate DixonDepartment of Molecular and Cellular Biology

http://www.childrensmuseum.org/teachers/unitsofstudy/biotechnology/lesson3.htm

5 mLight microscope resolving power ~0.2 m

Human cell DNA

6 x 109 base pairs

2 meters of DNA

4 x 105 times the nuclear diameter

Organization of DNA in the human cell nucleus

http://www.aw-bc.com/mathews/ch28/fi28p12.htm

Leffell and Brash, Scientific American, July 1996

Leffell and Brash, Scientific American, July 1996

2%-10% UVB reaches basal layer of skin

Midwest: 20 min sunlight would kill 50% of cells in culture dish

UV

Sun exposure – ultraviolet radiation

http://www.medicalecology.org/images/atmosphere/dimersfromp345.gif

Cellular Responses to DNA Damage

Apoptosis

DNA repair

Cell cycle delay

Mutagenesis

DNA damage

CANCER

http://www.genmapp.org/HTML_MAPPs/Human/Biological_process/response_to_DNA_damage_stimulus/_Support/response_to_DNA_damage_stimulus.jpg

Proteins Involved in Responses to DNA Damage

Genetic Alterations: Minimum requirements for transformation

RB1—Resistance to growth inhibition (more cell “birth”) TP53—Evasion of apoptosis (less cell “death”) RAS—Stimulation of cell proliferation TERT—Prevention of finite life span (“immortalization”)

Genetic Alterations in Cancer

Mut

atio

ns

Xeroderma pigmentosum patient showing distribution of skin lesions

Cancer

DNA damage

Repair

Mutation Normal

Xeroderma pigmentosum:

Extreme sun sensitivity, high risk of skin cancer

http://www.mdconsult.com/das/book/body/160721681-2/0/1492/I4-u1.0-B978-1-4160-2805-5..50467-5--f13.fig?tocnode=54631832

Goldman: Cecil Medicine, 23rd ed.

Copyright © 2007 Saunders, An Imprint of Elsevier

Nucleotide Excision Repair

Repair of UV-induced DNA damage

http://locus.umdnj.edu/nigms/pathways/ner.html

Cancer in xeroderma pigmentosum and related disorders of DNA repairJames E. CleaverNature Reviews Cancer 5, 564-573 (July 2005)doi:10.1038/nrc1652

Xermoderma pigmentosum variant:

High skin cancer risk, normal NER

DNA replication blocking lesions: UV photoproducts, DNA adducts, etc.

TTXPV is DNA polymerase

Can insert “A” opposite T in TT dimer – overcome replication block

In absence of XPV – other less accurate polymerase replicate past lesion

DNA repair

BER NER MMR

Mutagenesis

Base substitutions Frameshifts

DNA adducts Base modifications Base mismatches

DNA damage

Crosslink repair Deletions

DNA crosslinks

DSB repair (HR; NHEJ)

Deletions Rearrangements

DNA breaks

Replication fork blockage

Base substitutions Deletions Rearrangements

Post-replication repair

DNA Repair Pathways

Replication fork integrity and double strand break repair

TT TT

Replication fork “collapse”

DNA double strand breaks

Ionizing radiation,etc.

Chromosome instability syndromes

NBS1MRE11WRNBLM/RECQL3BRCA1, BRCA2ATM

Nijmegen breakage syndrome A-T-like disorderWerners syndromeBlooms syndromeFamilial breast cancerAtaxia telangiectasia

LymphomaLymphoma/leukemiaVariousLeukemia,carcinomasBreast/ovarian cancerLeukemia,lymphoma

Ataxia TelangiectasiaIncreased cancer risk Neurodegeneration Chromosomal instability Abnormal responses to UV and IR

IR: Chromosomal aberrations; Cell killingUV: Chromosomal aberrations

350 kDa serine/threonine protein kinase Related to ATR, DNA-PK, Mec1, Tel1

ATM Kinase

www.atcp.org

ApoptosisDNA repair

Cell cycle checkpoints

ATM

www.biocarta.com/pathfiles/atmPathway.asp

Error Free Error Prone

Recombination End Joining

Dividing Cells

Non-Dividing

Cells

StrandInvasion

DSB

Resection

DNA synthesisLigationJoint molecularresolution

DSB

End Binding

Ligation

A: HR B: NHEJ

DSB

Resection

C: MMEJ

pairing

Ligation

Replication fork integrity and double strand break repair

Chromosome instability syndromes

NBS1MRE11WRNBLM/RECQL3BRCA1, BRCA2ATM

Nijmegen breakage syndrome A-T-like disorderWerners syndromeBlooms syndromeFamilial breast cancerAtaxia telangiectasia

LymphomaLymphoma/leukemiaVariousLeukemia,carcinomasBreast/ovarian cancerLeukemia,lymphoma

MRN complex: Mre11, Rad50 and Nbs1

• Mre11: Nuclease (both endo- and exo-nuclease)

• Rad50: DNA binding

• Nbs1: Protein-protein interactions

Mre11/Rad50/Nbs1 (MRN) Complex

DNA double-strand break repair

http://www.eurekalert.org/features/doe/2002-11/dbnl-tsb110702.php

What is the function of ATM in non-dividing cells?

DSB

End Binding

Ligation

B: NHEJDSB

Resection

C: MMEJ

pairing

Ligation

ATM

In the absence of ATM (Ataxia Telangiectasia):

MMEJ is increased

End resection is increased

MRN

ATM

MRN

BRCA1

MRN

NHEJ: BRCA1 Inhibits MRN Nuclease by Binding to DNA Ends (Paull et al., 2001)

ATM

Promotion of DNA Binding

Inhibition of DNA degradation

Mre11 nuclease

?

HR: BRCA2 Regulates Homologous Recombination by Binding to Rad51

BRCA2

ATM

BRCA2P

Rad51Rad51

Free to promote recombination

BRCA1 and BRCA2 Promote Recombination Repair and Inhibit End Joining

Mitotic somatic cells

Post-Mitotic neuronal cells

Failure to repair or misrepair

Cancer Neurodegeneration

Cellular responses to DNA damage

ATM

Defective DNA Repair and Neurological DysfunctionDefective DNA Repair and Neurological Dysfunction

Rass, U. et al. 2007; Cell, 130: 991-1004.

http://www.genome.jp/dbget-bin/get_pathway?org_name=bsu&mapno=03430

Microsatellite sequences are repeated sequences that can easily misalign during DNA replication – causing changes in the length of the sequence. Normally these mistakes are corrected by MMR

Expansion of microsatellite sequences is often observed in tumor tissue from patients with hereditary nonpolyposis colorectal cancer (HNPCC; also called Lynch Syndrome)

http://www.bma.org.uk/health_promotion_ethics/genetics/Cancergenetics.jsp?page=12

http://www.ehponline.org/members/1997/Suppl-4/peltomaki-full.html

http://asajj.roswellpark.org/huberman/DNA_Repair/MMRproteins.gif