cellular response to dna damage - repair
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Cellular Response to DNA Damage - Repair. ENVR 430: Health Effects of Environmental Agents October 3, 2008 John R. Ridpath Rosenau 347 966-6141. DNA Background. DNA encodes all genetic information Original assumption: blue-print for life must be fundamentally stable - PowerPoint PPT PresentationTRANSCRIPT
Cellular Response to Cellular Response to DNA Damage - RepairDNA Damage - Repair
ENVR 430: Health Effects of Environmental AgentsOctober 3, 2008
John R. RidpathRosenau 347966-6141
DNA BackgroundDNA Background DNA encodes all genetic information Original assumption: blue-print for life must be
fundamentally stable Physicist Erwin Schrödinger (in his monograph
“What is Life”, 1944): suggested changes could occur to the “hereditary code script”
It was known x-rays could break chromosomes
Schrödinger said the lesions could be replaced by “ingenious crossings” with the unharmed chromosome – we now call this DNA repair mechanism homologous recombination
DNA primary structure elucidated in 1953
Terminology RemedialTerminology Remedial Mutation – heritable change in sequence of genome Mutant – organism that carries one or more
mutations Genotype – genetic information organism encodes
in its genome Phenotype – ensemble of observable characteristics
of an organism Mutagen – agent that leads to an increase in the
frequency of occurrence of mutations Mutagenesis – process by which mutations are
produced
DNA DamageDNA Damage
Our genome (primary structure of DNA) is continually beset with insults caused by a myriad of agents, both endogenous and exogenous to the cell.
After DNA Damage, then After DNA Damage, then What?What? Acute Long-Term
Cancer
Aging
Degenerative disease
Mutation
Cell death
DNA repair Healthy
Slide courtesy of Brian Pachkowski
Sources of DNA DamageSources of DNA Damage
Endogenous sources Spontaneous hydrolysis of bond between base and
sugar of backbone; 18000 purines (A & G)/cell/day lost
Deamination of cytosine to uracil; 100-500/cell/day Oxygen radicals (ROS) react with bases; Ex: 8-
oxoG, 1000-2000/cell/day Replication errors; enough errors to be devastating Methylating agents (Ex: SAM); react with all bases,
1200/cell/day
Sources of DNA DamageSources of DNA Damage
Exogenous sources Ionizing radiation; radioactives, cosmic rays Man-made chemicals react with and alter DNA
structure and chemistry UV radiation from sun; fuses adjacent bases
(thymine dimers)
Examples of DNA DamageExamples of DNA Damage
DNA RepairDNA Repair
DNA repair “…connote(s) cellular responses to DNA damage that result in the restoration of normal nucleotide (base) sequence and DNA structure…” *
* Friedberg, et al., DNA Repair and Mutagenesis, 2nd ed.; ASM Press; Washington, D.C., 2006; p 4.
DNA Repair PathwaysDNA Repair Pathways
Direct Direct reversalreversal MismatchMismatch
Base Base excision/excision/
SSBSSBNucleotide Nucleotide excisionexcision
Homologous Homologous recombinationrecombination
Non-Non-homologous homologous end joiningend joining
Type Type of of
LesionLesion
O6-MeGuanine,
Pyrimidine dimers
Mispaired
bases
Alkylations, Alkylations, oxidations, oxidations,
abasic abasic sites, sites, strand strand breaksbreaks
Bulky or helix
distorting adducts
Double strand breaks,
crosslinks
Double strand breaks
Slide courtesy of Brian Pachkowski
Direct Reversal of DNA Direct Reversal of DNA DamageDamage Repairs: pyrimidine dimers (UV), methylated bases How: enzymatic reaction – just changes it back
DNA methyltransferases: proteins that remove methyl groups from bases
Cryptochrome: human enzyme that reverses pyrimidine dimers
Fidelity: Most efficient, most accurate repair – single enzyme, single step
Consequence of failure: Dimers; interference with replication and transcription methylated bases; GC → AT transitions, heritable
mutations
Direct Reversal of DNA Direct Reversal of DNA DamageDamage
The proteins MGMT and ABH2 are used to directly remove methyl groups in direct reversal
Wyatt and Pittman, Chem. Res. Toxicol. 2006, 19, 1580-1594
Mismatch RepairMismatch Repair
Repairs: improperly paired nucleotides and insertion/deletion loops during replication
How searches for signal that identifies newly synthesized
strand; template strand contains methylated bases, new strand is not immediately methylated
degrades this strand past mismatch resynthesizes the excised strand
Consequences of failure: increased susceptibility to cancer, especially hereditary non-polyposis colorectal carcinoma (HNPCC)
Mismatch RepairMismatch Repair
GATC
G
GATC
G
5’
GATC
G
5’
3’GATC
G
5’
CTAGT
3’ Me
5’
3’5’
1. Enzyme complex recognizes G:T mismatch in hemimethylated DNA
2. Excises mismatched nucleotide (T) on unmethylated strand and reinserts correct nucleotide
Base Excision RepairBase Excision Repair When thine eye offends thee … Repairs
oxidized/reduced bases (Ex: 8-oxoG, 1000- 2000/cell/day)
alkylated bases deaminated bases mismatched bases (replication errors) missing bases [apurinic, apyrimidinic (AP)
sites] How: removes offending base and replaces with
correct base Fidelity: excellent
Base Excision RepairBase Excision Repair
Consequences of failure Base substitution → transitions, transversions →
point mutations AP sites Single strand breaks that may lead to double
strand breaks
Base Excision RepairBase Excision Repair
Adapted from Sancar, et al., Annu. Rev. Biochem. 2004. 73:39-85
Short patch
Long patch
Nucleotide Excision RepairNucleotide Excision Repair Repairs: cyclobutane pyrimidine dimers
(CPD), bulky adducts (i.e., B[a]P), AP sites, intercalated compounds, DNA interstrand crosslinks
How Recognition and verification of base damage Incision of DNA strand on either side of damage Excision of oligonucleotide fragment generated by
incisions Repair synthesis to fill the gap Ligation of nick in DNA
Nucleotide Excision RepairNucleotide Excision Repair
Fidelity: Excellent Consequences of failure
Interference with replication, transcription
Nucleotide Excision RepairNucleotide Excision Repair
Adapted from Sancar, et al., Annu. Rev. Biochem. 2004. 73:39-85
Recognition and verification of damageRecognition and verification of damage
Nucleotide Excision RepairNucleotide Excision RepairRecognition and verification of damageRecognition and verification of damage
Nucleotide Excision RepairNucleotide Excision RepairIncision on either flank of affected strandIncision on either flank of affected strand
Nucleotide Excision RepairNucleotide Excision Repair
PIC 4
Excision of affected oligonucleotide and resynthesis Excision of affected oligonucleotide and resynthesis of strandof strand
Nucleotide Excision RepairNucleotide Excision Repair
PIC 5
Ligation of nick in DNA strand by DNA ligase I (not Ligation of nick in DNA strand by DNA ligase I (not specifically shown)specifically shown)
Double Strand Break RepairDouble Strand Break Repair Two types of DSB repair
Homologous recombination (HR) Non-homologous end joining (NHEJ)
DSB Caused by: ionizing radiation/ROS, replication fork encountering single-strand break, other repair mechanisms
Experimental evidence suggests NHEJ is the primary mechanism used early in the cell cycle (G1) while HR is used later (S/G2)
Double Strand Break RepairDouble Strand Break Repair
Consequences of failure Sister chromatid exchanges (SCE) Aneuploidy – loss or duplication of
chromosomes or chromosomal segments (proposed as the initiating event for cancer)
Double Strand Break RepairDouble Strand Break RepairHomologous RecombinationHomologous Recombination Repairs: DNA double-strand breaks How
Utilizes another DNA molecule that has a similar (homologous) or identical DNA sequence (sister chromatid)
One strand on each side of the break in the damaged molecule is degraded to leave 3’ single strands
One of the single strands then invades the homologous nucleotide sequence of the other DNA molecule using it as a template to reconstruct the damaged molecule
Fidelity: Virtually error free, especially if sister chromatid is used
Double-strand Break Repair by Homologous Recombination
Slide courtesy of Jeff Sekelsky
Damage removal, resection
strandinvasion
XXDSB
Displaced yellow strand iscaptured by blue strand
Homologous DNA strand Crossovers (Holliday junctions) are then resolved
Double Strand Break RepairDouble Strand Break RepairNon-homologous end joiningNon-homologous end joining
Double strand break repair the easy way – just deal with it
How Protect and trim the ragged ends Bridge the gap Ligate the nicks
Fidelity: poor – deletions can result in loss of coding information
Non-homologous End JoiningNon-homologous End Joining
Adapted from Sancar, et al., Annu. Rev. Biochem. 2004. 73:39-85
Examples of Human Genetic Diseases Examples of Human Genetic Diseases
Caused by Dysfunctional Repair PathwaysCaused by Dysfunctional Repair Pathways Human disease
Gene(s) Defective
pathway
Clinical
featuresXeroderma
pigmentosum (XP)XPA-XPG; XPV NER Dermatitis, skin cancer,
neurological defects
Nijmegen breakage
syndrome (NBS)
NBS1 Strand break repair
Developmental abnormalities growth retardation, cancer
predisposition
Cancer BRCA1,BRCA2 HR Hereditary breast, ovarian
cancer Fanconi anemia FANCs,BRCA2 HR Limb defects, anemia,
cancer
Hereditary non-polyposis colon
cancer (HNPCC)
MSH2, MSH3,
MSH6, others
Mismatch repair
Colon and other cancers
Single Nucleotide Single Nucleotide Polymorphisms (SNP)Polymorphisms (SNP) SNP: a change in a single
nucleotide on one allele when a gene on both alleles is compared
Occurrence in human genome: approximately one in every ~1330 bases
An allele is defined as polymorphic if it appears in > 1% of the population
Can alter protein function including that of repair proteins (Ex: XRCC1 used in BER)
DNA strand 1 differs from DNA strand 2 at a single base-pair location (a C/T polymorphism).
Mutator PhenotypeMutator Phenotype
Most cancer cells exhibit greater numbers of mutations than would be expected randomly
Mutator phenotype: results from mutations in genes that are responsible for genomic stability (i.e., genes for repair proteins, genes responsible for the proper segregation of chromosomes during mitosis)
Allows for accumulation of massive numbers of mutations
Can have a cascade effect if even more repair proteins become mutated