DNA Damage and Repair · 2015. 9. 3. · DNA Damage and Repair • Our genome contains the information necessary for proper cell function and survival • Mutations can lead to reduced

Skin Biology Lecture Series

DNA Damage and Repair


•  Our genome contains the information necessary for proper cell function and survival

•  Mutations can lead to reduced function, cancer, or death

•  Protection from DNA damage and repair of DNA damage is critical to function and survival

•  We are exposed to potential DNA damage each day Source: Wolff, Goldsmith, Katz, Gilchrest, Paller, Leffell. Fitzpatrick’s Dermatology in General Medicine, 8thEd. Copyright The McGraw-Hill Companies. All rights reserved. Figure e112-1.1.

• Sources of DNA Damage

• Exogenous • Reactive oxygen species •  Ultraviolet Light •  Chemical carcinogens

•  Endogenous • Aerobic metabolism- oxidative damage •  Spontaneous mutations


Source: openclipart.org

Ultraviolet Radiation-Induced DNA Damage

Source: Wolff, Goldsmith, Katz, Gilchrest, Paller, Leffell. Fitzpatrick’s Dermatology in General Medicine, 8thEd. Copyright The McGraw-Hill Companies. All rights reserved. Figure 112-1.

Ultraviolet DNA Damage: CPD dimers



Ultraviolet DNA Damage

• UVB also leads to 6-4 photoproducts • UVA and UVB lead to oxidative damage, formation of 8-hydroxy-deoxyguanosine products

•  Nucleotide excision repair (NER) is the primary repair mechanism for UVR-induced cyclobutane dimers and (6-4) photoproducts and damage induced by certain chemical carcinogens

•  NER pathways include:

•  global genome repair (GGR) operates across global genes •  transcription coupled repair (TCR) operates on actively

transcribed genes; 10% of genes are actively transcribed at any point in time

DNA Repair: Nucleotide Excision Repair


•  In GGR, DNA photoproducts recognized by XPC or XPE bound to HHRAD23B (R23) HR23 proteins

•  In TCR, RNA polymerase detects damaged DNA, recruits CSA and CSB

•  Several other proteins then bind: •  XPA and RPA aid in

recognition of base damage

•  TFIIH helicase unwinds helix, is composed of six subunits including XPB and XPD

•  TFIIH allows binding of polymerase


DNA Repair: Nucleotide Excision Repair


DNA Repair: Nucleotide Excision Repair •  ERCC1 and XPF bind to repair complex • XPG endonuclease cuts damaged strand at the 3’ junction between single and double strands • ERCC1 and XPF endonuclease cut damaged strand at 5’ junction • Olignucleotide fragment ~ 27-30 nucleotides in length which includes the damage is excised •  27-30 nucleotide gap replaced by repair synthesis • Repair synthesis requires DNA polymerase (POLδ/ε) and replication proteins PCNA, RPA and RFC

Nucleotide Excision Repair

•  PCNA protein acts as a DNA clamp and processivity factor for DNA polymerase delta

•  Processivity is ability of an enzyme to continue catalysis without dissociating from substrate

•  Processivity of DNA polymerase enzymes correlates with the average number of nucleotides that can be added

•  DNA polymerases associated with DNA replication usually highly processive; those associated with DNA repair usually low processivity

•  Replication protein A (RPA): three-subunit single-stranded DNA-binding protein

•  Replication factor C (RFC) binds to the 3' end of new DNA strand and loads PCNA and DNA polymerase delta onto template

•  POLd synthesizes the homologous DNA strand •  Ligase connects the two ends Source: Wolff, Goldsmith, Katz, Gilchrest, Paller, Leffell. Fitzpatrick’s Dermatology in General Medicine, 8thEd. Copyright The McGraw-Hill Companies. All rights reserved. Figure 110-3.

In which condition is there compromised Global genome repair (GGR)?


In which condition is there compromised Global genome repair (GGR)?

Xeroderma Pigmentosum (XP)

•  Autosomal recessive disease •  photosensitivity, pigmentary changes, premature skin aging, skin

cancer development •  Defect in nucleotide excision repair (NER) •  Seven xeroderma pigmentosum repair genes, XPA through XPG

identified

NER Defects: Xeroderma pigmentosum and Cockayne syndrome

•  Xeroderma pigmentosum: •  photosensitivity •  poikiloderma (hyper- and hypo-pigmentation, atrophy,

telangiectasia) •  skin cancers: BCC, SCC, melanoma •  central nervous system tumors •  sensorineural deafness, progressive neurologic degeneration,

primary loss of neurons (some patients) •  Cockayne syndrome (CS):

•  Photosensitivity •  no increased incidence of cutaneous neoplasia •  Typical facial features (deep-set eyes, loss of subcutaneous fat) •  pigmentary retinal degeneration •  post-natal growth failure, sensorineural deafness, progressive

neurologic degeneration, primary dysmyelination, brain calcifications

Source: Bickers et al. Oxidative Stress in the Pathogenesis of Skin Disease. Journal of Investigative Dermatology (2006) 126, 2565–75.

Oxidative DNA Damage

•  UVA induces reactive oxygen species formation

•  Aerobic metabolism and other exogenous ROS sources also lead to oxidative damage

•  Oxidative damage leads to formation of 8-oxo-guanine

•  DNA damage and inflammation result

DNA Repair: Base Excision Repair

Source: Wolff, Goldsmith, Katz, Gilchrest, Paller, Leffell. Fitzpatrick’s Dermatology in General Medicine, 8thEd. Copyright The McGraw-Hill Companies. All rights reserved. Figure e112-1.1.

•  Mismatch base substitutions occur during DNA replication and recombination •  Repair enzymes recognize incorrect base:

•  Template strand is methylated •  Newly synthesized strand is not yet methylated yet

•  MutS binds mismatched base pairs •  MutL is recruited and activates MutH •  MutH cleaves unmethylated strand •  Mismatch segment removed by exonuclease and helicase II •  Gap is filled by DNA polymerase III and DNA ligase

DNA Repair: Mismatch Repair

Source: Young et al. DNA Mismatch Repair Proteins: Potential Guardians Against Genomic Instability and Tumorigenesis Induced by Ultraviolet Photoproducts. JID (2003) 121: 435-440.

Which dermatologic syndrome has defective mismatch repair?


Which dermatologic syndrome has defective mismatch repair?

Muir-Torre Syndrome

•  Signaling cascades activated in response to DNA damage •  If mild damage, cells often undergo cell cycle arrest •  If severe damage, cells often undergo apoptosis


Cellular Signaling After Mutation


Cellular Signaling After Mutation

In which syndrome are there germline mutations in the gene that

encodes p95/Nbs1?


•  Nijmegen breakage syndrome •  Autosomal recessive •  chromosomal instability •  microcephaly •  distinct facial appearance •  short stature •  immunodeficiency •  radiation sensitivity •  increased risk of lymphoid malignancy

In which syndrome are there germline mutations in the gene that

encodes p95/Nbs1?

Homologous Recombination Repair

A patient with Fanconi’s anemia, telangiectasias, poikiloderma and cutaneous SCCs. Source: Kamenisch et al. Progerioid syndromes and UV-Induced oxidative DNA damage. Journal of Investigative Dermatology Symposium Proceedings (2009) 14, 8–14; doi:10.1038/jidsymp.2009.6

•  Non-homologous end joining (NHEJ): Ligation of two DNA ends with little or no homology to one another

•  Double strand breaks can lead to joining of DNA ends on two different chromosomes. This is error-prone and is frequently associated with deletions and translocations

•  DNA end-binding complex XRCC5 (Ku80) and XRCC6 (Ku70) helicases are activated by phosphorylation

•  DNA-PK phosphorylates Ku70 and Ku80 •  XRCC4 mediates binding of ligase IV to the broken DNA ends •  Ligase IV joins the DNA ends

Non-homologous End Joining

Source: Mazarelli et al. Differential Modulation of Ku70/80 DNA-Binding Activity in a Patient with Multiple Basal Cell Carcinomas. Journal of Investigative Dermatology (2003) 121, 628–633; doi:10.1046/j.1523-1747.2003.12416.x

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DNA Damage and Repair · 2015. 9. 3. · DNA Damage and Repair • Our genome contains the information necessary for proper cell function and survival • Mutations can lead to reduced