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