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Skin Biology Lecture Series
DNA Damage and Repair
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
DNA Damage and Repair
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
Source: Wolff, Goldsmith, Katz, Gilchrest, Paller, Leffell. Fitzpatrick’s Dermatology in General Medicine, 8thEd. Copyright The McGraw-Hill Companies. All rights reserved. Figure 110-2.
Source: Wolff, Goldsmith, Katz, Gilchrest, Paller, Leffell. Fitzpatrick’s Dermatology in General Medicine, 8thEd. Copyright The McGraw-Hill Companies. All rights reserved. Figure 112-2.
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
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 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
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.
DNA Repair: Nucleotide 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 110-3.
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)?
Source: Wolff, Goldsmith, Katz, Gilchrest, Paller, Leffell. Fitzpatrick’s Dermatology in General Medicine, 8thEd. Copyright The McGraw-Hill Companies. All rights reserved. Figure 139-1.
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?
Source: Wolff, Goldsmith, Katz, Gilchrest, Paller, Leffell. Fitzpatrick’s Dermatology in General Medicine, 8thEd. Copyright The McGraw-Hill Companies. All rights reserved. Figure 119-2.
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
Source: Wolff, Goldsmith, Katz, Gilchrest, Paller, Leffell. Fitzpatrick’s Dermatology in General Medicine, 8thEd. Copyright The McGraw-Hill Companies. All rights reserved. Figure 110-4.
Cellular Signaling After Mutation
Source: Wolff, Goldsmith, Katz, Gilchrest, Paller, Leffell. Fitzpatrick’s Dermatology in General Medicine, 8thEd. Copyright The McGraw-Hill Companies. All rights reserved. Figure 110-4.
Cellular Signaling After Mutation
In which syndrome are there germline mutations in the gene that
encodes p95/Nbs1?
Source: Wolff, Goldsmith, Katz, Gilchrest, Paller, Leffell. Fitzpatrick’s Dermatology in General Medicine, 8thEd. Copyright The McGraw-Hill Companies. All rights reserved. Figure 110-4.
• 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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