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Cellular Responses to DNA Damage

-wide range of environmental factors and normal metabolic process can damage nearly 1 million nucleotides per cell per day
-lesions must be repaired if cell is to function normally
-every cell employs several mechanisms to repair damage before it's permanently encoded into genome and transmitted to daughter cells


Sources of DNA Damage We Discuss

-alkylation and hydrolysis of bases
-formation of nucleotide dimers by fusion of adjacent baces
-formation of either single or double stranded breaks


Repair of Damaged Nucleotides

-nucleotides that are damaged by environmental factors and normal metabolic processes are removed and replaced by base-excision and nucleotide-excision enzymes
-these two sets of enzymes will not attempt to directly reverse the damage process
-instead, systems will simply remove damaged base and replace it with newly synthesized DNA


DNA photolyase and Guanine Methyltransferases

-work on damaged nucleotides and directly reverse the DNA damage
-several error prone polymerases play important roles in bypassing deleterious effects of some damaged bases


Oxidation of Nucleotides

-oxidative damage is serious problem for cell
-ex: oxidation of guanine
-normally these two sets of enzymes will not attempt to directly reverse the damage process
-byproducts of normal metabolic processes can lead to creation of enol and keto forms of guanine
-keto tautomer of guanine can still form a base pair with cytosine but it can now also form a stable base pair with adenine (G:A).
-if not corrected G:C pair will be converted into A:T pair in subsequent rounds of replication
-fif G:A pair is located within the core promote or enhancer element this could disrupt transcriptional regulation
-likewise, if the G:A pair is located within an exon this could alter the structure and inactivate the encoded protein


Deaminition of Nucleotides:

-nucleotide bases are subject to deamination reactions through several different mechanism
-as DNA resides in an aqueous environment, deamination can occur through spontaneous reactions with water molecules
-can also also occur if DNA comes in contact with nitrous acid (HNO2)
-finally, the cell contains enzymes whose role is to deaminate nucleotides under special circumstances
-regardless of mechanism
-deamination occurs much more frequently than can be tolerated and must be corrected
-ex: deamination of cytosine results in formation of uracil, a base similar in structure to cytosine but used primarily in RNA
-uracil can hydrogen bond with adenine thus G:C pair can be converted into A:U pair
-if cytosine nucleotide resides within coding exon, it's possible for this to direct a change in amino acid composition


Base Excision Pathway

-the primary route for bases damaged by deamination, oxidation and hydrolysis to be removed and replaced
-enzymes that function within this cascade are constantly surveying the genome for physical distortions, incorrect base pairing and inappropriate modified nucleotides
-once damaged base has been recognized the base is excised by a generic glycosylase enzyme which will result in formation of apurinic or apyrmidinic site (phosphate+sugar)
-set of exo and endonucleases will then come in and make single stranded break around AP site
-DNA polymerase will then replace the base using the undamaged strand as the template
-finally DNA ligase will make the last phosphodiester bond.


Execution of Base Excision in Organisms

--executed in different ways in different organisms
-some species remove neighboring nucleotide along with damaged one
-other species activities of glycosylase and AP exo/endonucleases are combined into one protein that will carry out both functions (neofunctionalization or subfunctionalization)


Comparison of Repair Mechanisms

-Mismatch Repair System is tasked with removing bases that were incorrectly added by DNA polymerase
-in contrast role of the Base Excision Repair System is to remove damaged bases and replace them with correct/intact bases
-Mismatch Repair System is used primarily during the replication process itself as the differences between parental and new DNA strands are the most evident
-Base Excision Repair System is used throughout the life of the cell to correct damage to nucleotides that are caused by routine metabolic processes and environmental factors
-Base Excision Pathway most often will remove just the nucleotide that is damaged (some occasions in which two adjacent nucleotides are removed)
-this system will remove bases from either the parental or newly synthesized strand
-Mismatch Repair system removes considerably more DNA – approximately 256bp on average
-also it only removes nucelotides from newly synthesized strands


Role of Fail-Safe DNA Glycosylases

-extraordinarily high number of damaged nucleotides that need to be removed in a cell every day back-up systems have evolved to ensure that damaged nucleotides that have not been removed by the Base Excision System do not get permanently fixed within the genome
-similar systems have evolved to ensure the mistakes that get past the proofreading activity of DNA polymerase are reversed prior to the next replication cycle
-in case of damaged nucleotides a panel of DNA glycosylase (fail-safe glycosylases) scan the genome looking for bases that have been overlooked by the Base Excision System
-designed to identify the different forms of damage nucleotides
-ex: guanine base that has undergone oxidation is considered. If this base is not removed by Base Excision enzymes then an adenine residue (instead of cytosine) will be incorporated within the new strand during DNA replication
-fail-safe DNA glycosylases will recognize the G:A pair, excise the adenine residue from the newly synthesized strand, and will replace it with the correct cytosine base
-restores the proper G:C pair and give the cell another opportunity to remove the damaged guanine nucleotide via the Base Excision Pathway


Nucleotide Excision Repair System

-nucleotides can also be modified such that physical distortions are induced within the DNA duplex
-modifications include the formation of thymine dimers in response to exposure to ultraviolet radiation and contact with organic compounds
-both types of modifications cause significant enough distortions that DNA polymerase (and RNA polymerase) will stall at the site of the modification
-if uncorrected the stalling of these polymerases will result in S phase arrest and/or inhibition of transcription
-modified nucleotides are recognized and removed by the Nucleotide Excision Repair system


Nucleotide Excision Repair Enzymes

-similar to the enzymes of the Base Excision Repair System, the Nucleotide Excision Repair System constantly surveys the genome throughout the entire life of the cell


Uvr-A, Uvr-B, and Uvr-C

-in bacteria complex containing these proteins (A and B) recognizes and binds to regions of distorted DNA
-these two enzymes will locally separate the two DNA strands and will recruit the Uvr-C nuclease which makes single stranded nicks on both sides of distortion (Mismatch Repair System makes single nick)
-nuclease will discriminate between the two strands and will only remove nucleotides on the strand the contains the damaged base


Uvr-D helicase

-recruited to unwind double helix
-since DNA strand nicked on both sides of modified base, unwinding of helix by Uvr-D sufficient to remove section of DNA strand without use of endonucleases
-small stretch of DNA removed


DNA polymerase and ligase

-fills gap created and strand is sealed by DNA ligase
-fill and seal all gaps created by removal of primers and removal of damaged bases and removal of short stretches of DNA containing either mismatches or physical distortions


Missed Distortion by Nucleotide Excision Repair

-if missed distorted base, will be caught during transcription when RNA polymerase stalls at the distortion site
-NER enzymes will be recruited by the polymerase thus providing the cell with another opportunity to correct this error


Error-Prone Polymerases

-if replication machinery encounters a thymine dimer, the polymerase will first stall and will then disassociate from the DNA
-error-prone polymerase will then be recruited to the site of the dimer because this polymerase is designed to make mistakes it will be able to incorporate nucleotides opposite the two thymine residues
-instead of adding two adenine residues it will add any combination of two bases
-results in the addition of incorrect bases it may be better then having replication stop completely
-not restricted to reading past thymine dimers and can read past other types of molecular lesions


DNA Photolyase Removal of Thymine Dimers

-adjacent thymine residues can be fused together to form dimers by exposure to ultraviolet radiation
-causes a distortion in the DNA double helix which in turn results in S phase arrest due to the pausing of DNA polymerase or inhibition of transcription due to the stalling of RNA polymerase
-Nucleotide Excision Repair enzymes recognize the physical distortions that are caused by the formation of thymine dimers and excises these bases
-thymine dimer formation occurs at a high frequency additional mechanisms are employed by the cell to correct these defects
-one system uses DNA Photolyase which directly reverses formation of thymine dimers by physically separating two thymine residues
-binds to thymine dimers but requires visible light to activate protein and separate two residues


Removing Methylated Nucleotides

-methylation of cytosine nucleotides plays an important role in transcriptional activation
-methylation of certain bases such as guanine can be harmful as the methylated base will adopt a new structure and may base pair with the wrong nucleotide
-ex: DNA polymerase will mistake methylated guanine for adenine and will incorporate a thymine nucleotide in the newly synthesized strand
-if uncorrected this G:T pair will be permanently encoded as G:T and A:T pairs in the daughter duplexes
-methyl group too small to cause distortion that can be recognized by nucleotide excision repair system
-methyl group removed by enzyme called methyltransferase
-with this mechanism the modified base is directly fixed without the need for the excision of any nucleotides


Ames Test

-is used to determine if a chemical can induce mutations within DNA
-strain of the Salmonella bacteria that contains a point mutation in a gene required for synthesis of the amino acid histidine is added to media that lacks the amino acid histidine (media has all other required nutrients)
-histidine is required for many proteins, its absence leads to defects in protein production
-bacterial cells cannot survive without histidine thus no colonies will grow on the plate
-same strain of Salmonella is then treated with the chemical in question
-if colonies appear on the plate you can conclude that mutations were induced that reverted the mutated gene back to wild type, which then allowed the cells to grow
-if no colonies appear on the plate then the chemical is not considered a mutagen
-if colonies appear on the plate then the chemical is not considered a mutagen


How can mutations occur?

-mutations can occur through exposure to environmental factors such as ultraviolet light and ionizing radiation
-for chemical to called a mutagen it must induce mutations at a higher rate than the background environment