Molecular Bio Lecture 9

endogenous; hydrolysis; oxidation; alkylation; exogenous; UV; ionizing; thymine

DNA is constantly subject to many_______sources of damage. These include spontaneous hydrolysis, ______and_________reactions at multiple positions.

________sources of damage including _____and_______radiation and various chemicals can lead to “bulky lesions” such as________dimers and chemical adducts and well as single and double strand breaks.

replicases; base configuration; tautomerization; indel

Despite the high fidelity of the cellular_______, nucleotide mis-incorporations occur on a regular basis due to the large size of genomes. These errors are exacerbated by________and_______. Replicase slippage can also produce______mutations. Some forms of damage associated with “genomic instability” can result in structural changes to entire chromosomes such as inversions, deletions, and translocations.

death; mutation; nothing; DNA replication

DNA damage can lead to three outcomes based on the level and type: ______,______, or_______. DNA damage does not automatically result in mutation. If the damage is repaired prior to________, no mutation will result.

common lesions; direct; base excision repair; nucleotide excision repair; mismatch repair; homologous recombination; non-homologous end joining

Cells encode multiple, energy-intensive repair pathways, some specialized for a single type of damage and others with broad and overlapping functions.________such as thymine dimers can typically be repaired by multiple pathways. Repair systems and the type of damage they address include _________repair (thymine dimers and certain types of base damage), __________(base damage), __________- (bulky lesions), __________ (mis-incorporation and small indels), and__________ and __________ (double-strand breaks). Specialized lower fidelity or “error-prone” polymerases can also contribute to DNA repair.

sensors; transducers; effectors; cell cycle; transcriptional; apoptosis

The global cellular response to DNA assault is highly organized. It includes _________that detect the damage, ________that signal the damage and ________that repair the damage and/or lead to other cellular outcomes such as________arrest, altered________-patterns or________(programmed cell death). Cells generally opt for the most conservative course resulting in the fewest number of mutations possible.

covalent; hydrolysis; cytosine; hydrolysis; 5-methylcytosine; thymine; hypoxanthine; N-glycosidic; abasic

Base Damage: Hydrolysis

All biochemical reactions take place in aqueous solution, so obviously water is always present at high concentration. Many of the______bonds in DNA are subject to spontaneous_______.

Deamination: The most common form of damage is deamination of _______residues to form_______, which occurs between 100-500 times per cell per day. Deamination of ___________generates a ________, which is particularly problematic since thymine occurs normally in DNA. Adenine residues can also be deaminated to _________, but this occurs less frequently.

Hydrolysis of the ________bond linking the base to the deoxy-ribose sugar generates an________site, and hydrolysis of the phosphodiester backbone leaves a nick in the DNA.

damage; mutation

Damage to Mutation:

•DNA damage and mutation are not the same thing.

•The cell has time to repair the_______before a_______becomes fixed in the DNA sequence.

point; transition; transversion

Types of _______Mutations = changes to a single nucleotide

•A________exchanges one purine for another one or one pyrimidine for another.

•A_______changes a purine for a pyrimidine or vice versa.

reactive oxygen species; exogenous

Oxidation and Alkylation:

__________which are a normal part of aerobic metabolism are an important source of DNA damage, as are________chemicals like those in cigarette smoke and industrial toxins. The Ames test is used to determine the genotoxic (DNA damaging) effects of chemicals.

nitrous acid

Deamination (again): In addition to simple hydrolysis, deamination of cytosine and adenine can be catalyzed by__________ (HNO₂); hypoxanthine can be recognized as something to get rid of

reactive oxygen species; 8-oxoguanine

Oxidation: All of the bases can be modified by_________. Oxidation of guanine to produce_________is particularly mutagenic. Most of nucleotides in anti-configuration —> preferred for steric reasons; everything out of way of everything else (Favored for normal guanine)

methylation; purines; bulky lesions; replication fork

Alkylation: Spontaneous, non-enzymatic_____of_______, particularly guanine, occurs frequently. The addition of larger alkyl groups, such as benzo[a]pyrene from cigarette smoke leads to________in the DNA that block_________ progression

syn; anti

Base alterations that favor the______configuration can lead to non-canonical base pairing considering that the_______configuration is the preferred configuration

isomers; keto-enol; amino-imino

Base Tautomerization:

•Tautomers are structural______with the same chemical formula

•_____–____and ____–_____tautomerization is common in nucleotide bases

enol; mutation

_______Base Tautomers form Different Base Pairs

Rare base tautomers can form non-canonical base pairs, allowing for their misincorporation during replication.

This is not really “damage” but it can lead to______if not repaired

cross-links; replication; transcription

Covalent________between adjacent (intra-strand) or opposite (inter-strand) bases represent an absolute barrier to DNA_______as well as ______. Many chemicals used in chemotherapy act in this manner and so preferentially target cells undergoing active DNA replication.

pyrimidine; thymine; dimers; phosphodiester backbone; double strand

Radiation:

UV: Most terrestrial organisms are subject to regular UV irradiation due to sun exposure. DNA absorbs in the UV range, and the energy is converted to covalent bonds between adjacent_______, most often_____, residues generating______that distort the DNA strand and are unable to base pair. A single unrepaired thymine dimer is sufficient to kill an E. coli cell, so numerous pathways exist to deal with them.

Ionizing radiation: Ionizing radiation, such as that emitted from radioactive chemicals, introduces nicks in the__________, which can lead to________breaks when they are clustered together. High levels of radiation can rapidly overwhelm the DNA damage response and kill most mammals, including humans, due to bone marrow failure and massive cell death in many tissues. When high levels of radiation are used therapeutically, they are followed by bone marrow replacement (transplant) which rescues the patient from an otherwise lethal dose.

structural; indels; inversions; translocations; reciprocal; non-reciprocal

________mutations include insertions, deletions, inversions and translocations.

a)   _________are small insertions and deletions.

b)   ________change the orientation of segments of chromosome.

c)   __________are the largest structural mutations. They include the _______or _____________movement of chromosome segments between non-homologous chromosomes. Frequent translocation is the hallmark of genomic instability.

triplet expansion; microsatellites

Indels: small insertions or deletions. Linked with regions where there are repeats; ___________diseases = normal range for them and for those with disease, the number of indels is much higher

Regions of repeats = _________—> become unstable and pretty common way that people identify and analyze blood

replication; transcription

Bulky Adducts: Cyclobutyl-thymine Dimers:

•Bulky adducts block ________and _________. They are an existential threat to the cell.

•Unrepaired, they can kill the cell outright. Mutations can occur during the removal and repair of these adducts.

replisome; base damage; damaged; mis-incorporated; dimers; adducts; crosslinks

DNA Damage and Mutation:

The primary problem with base damage is that altered bases form non-standard base pairs, and the damaged bases are usually not obstacles for the_________. However,_________ (or nucleotide mis-incorporation) does not automatically result in mutation. DNA replication using the __________or__________base as a template is necessary to establish the mutation in the DNA strand. If the damage is repaired prior to replication, or in some cases after only one round of replication, no mutation will result.

Bulky DNA lesions including pyrimidine______, large base________, and strand_______block DNA replication. If left unrepaired, they will kill the cell outright. Repair processes can actually bring about structural mutation in these cases, most often through recombination errors.

crosslinks; replication; transcription

Bulky Adducts: Chemo Agents

Chemo agents like cisplatin can generate intra-or inter-strand________. Both will block________and__________

single-strand; double strand breaks

Ionizing Radiation: DSBs

•Ionizing radiation causes_______nicks that tend to cluster.

•Multiples clustered nicks lead to gaps and___________

•DSB can occur accidentally during multiple repair pathways, so any type of damage can potentially generate a DSB.

unrepaired; surveillance; deletion; duplication

Large scale mutations are the end result of _________DSB coupled with a breakdown of cellular_______. Damage that is visible under microscope. _________of entire region of chromosome or_______; part of one chromosome and combine with another

sensor; transducer; effectors; template; opposite strand; sister chromatid

Principles of DNA Repair:

Several things are required for successful repair of DNA damage. The cell must have a way of recognizing the damage, the job of______proteins. The presence of damage must then be signaled by_______proteins to_________that actually repair it. In some cases, all of these activities are combined in a single protein, but in other the system is enormously complex and is fully integrated with all other cellular functions (e.g. transcription, translation, cell cycle progression and DNA replication). Nearly all forms of DNA repair require an intact________such as the ________of DNA or the_________.

thymine dimers; photolyase; placental mammals; O6-methyl-guanine methyltransferase

Direct Repair:

The most common lesions can be directly repaired in certain organisms. This is in contrast to repair systems that must remove the damage and replace it.

1)________. ________is an enzyme that uses visible light to repair thymine dimers, converting them back to separate thymine residues. This enzyme is present in nearly all organisms but is absent in_________, which have developed an alternative pathway for high fidelity repair of thymine dimers (discussed below).

2)   O₆-methyl-guanine.___________catalyzes the demethylation of O₆-me-G, converting it back to intact guanine. Each molecule of O₆-methyl-guanine methyltransferase can be used only once, and cells expend many of these molecules every day solely for the purpose of repairing this single type of base lesion.

O6-meG-methyltransferase; photoreactivation

Damage	System	Mutant Phenotypes/Diseases
O6-methylguanine O6-ethylguanine	Direct—_______	Mgmt-/- mice display hypersensitivity and tumorigenesis in response to alkylating agents
Thymine Dimers	Direct—_________	Not present in placental mammals

base excision repair; glycosylases; abasic; AP endonuclease; polymerase I; ligase; short patch; long patch; low; high; Fen1; Lig1

Other types of base damage, such as cytosine deamination, are addressed by the________system. Cells encode a battery of ___________, each of which recognize and excise particular damaged bases, leaving an_______site in the DNA. For particularly common lesions (e.g. 8-oxo-guanine and uracil), there may be multiple glycoslases devoted just to that single lesion, each recognizing it under different circumstances. This abasic site is recognized by an__________ (AP stands for apurinic or apyrimidinic), which nicks the DNA.

1)   In E. coli, the repair is completed DNA_______and______.

2)   In eukaryotes, the repair may be completed by either the______ or _______pathway.

a)   In short patch, the gap in filled by a relatively _____fidelity polymerase, Polß, and sealed by XRCC1/Lig3. Since only a single nucleotide is incorporated, the chance of mutation is low.

b)   In long patch, _______fidelity Pol∂e is recruited to carry out a longer region of displacement synthesis. The flap generated by this process is removed by_______and the nick is sealed by ____.

base excision repair; nucleotide excision repair; transcription-coupled; mismatch

Damage	System	Mutant Phenotypes/Diseases
Abnormal bases (e.g. uracil) Alkylated bases Oxidized bases	__________ (BER)	Ogg-/-Myh-/- mice have marked tumor predisposition (lung ovary, lymphoma, small intestine) hMYH alleles associated with colorectal tumors
Pyrimidine dimers Bulky Lesions Intra-strand crosslinks	_______(NER) __________Repair	Xeroderma Pigmentosum (XP): extreme sun sensitivity, 2000X increase in skin cancers, 10-20X increase in other cancers Cockayne syndrome (CS): hypersensitivity to UV radiation, stunted growth, neurological dysfunction, normal skin cancer rates
Mis-incorporated bases Small Indels	________(MMR)	Hereditary nonpolyposis colorectal cancer (HNPCC, actually affects several organs) Microsatellite instability, “Mutator” phenotype

glycosylases; replication fork; RNA polymerase; sensor; deformation; stalled; transcription coupled repair; excinuclease; helicase

Nucleotide Excision Repair:

Larger lesions that cannot be recognized by______and that block progression of the__________ or the________—such as thymine dimers and cisplatin crosslinks—are removed at the whole nucleotide level.

Unlike BER, this system does not have to recognize the specific type of damage; any bulky lesion can be excised and a region of DNA on ether side of the lesion is also removed. The system relies on a_______protein that recognizes a _________in the DNA, or it can be activated by a _________RNA polymerase in_________ (cells have a tendency to preferentially repair the template strand at genes undergoing transcription). An __________(a specialized type of endonuclease) is then recruited to nick the DNA on either side of the lesion, the piece of single stranded DNA including the lesion is removed by a_______, and the gap is filled by a DNA polymerase.

UvrABC excinuclease; sensor; nicks; DNA pol I; RNA polymerase; MFD transcription-coupled factor

E. coli NER. The__________ is critical for this process. DNA distortions are recognized by the UvrA_______protein, which recruits UvrB to coordinate the repair response. UvrB recruits UvrC, which displaces UvrA and introduces______five bases downstream and eight bases upstream of the lesion. The 13 bp segment is removed by UvrD, and the gap is filled by_______. When the __________ stalls at a lesion in the template, the__________is recruited to mark the lesion. This then leads to rapid recruitment of the UvrABC proteins.

xeroderma pigmentosum; cockayne syndrome; global genome repair; excinucleases; polymerase; transcription coupled repair

Mammalian NER. Many of the proteins associated with mammalian NER have been identified based on their association with two hereditary diseases, ________(XP) and ________(CS). XP is associated with extreme sensitivity to sunlight (photosensitivity) and cancer predisposition (particularly skin cancer), while CS is associated with growth failure, neurological dysfunction, premature aging and problems with many organ systems.

a) _________ (GGR). DNA lesions are recognized by the damage DNA binding protein (DDB), which recruits XP-C, which in turn recruits additional XP proteins as well as the TFII-H complex (which we will learn later is a basal transcription factor). After duplex opening by components of this complex, nicks are introduced on either side of the lesion by the XP-F and XP-G _________. The gap resulting from removal of the lesion is filled by a high fidelity________. The extreme photosensitivity of XP patients is due to the importance of NER in repair of thymine dimers.

b)   __________. When RNA Pol II stalls at a lesion, the CS-A and CS-B proteins are recruited in a manner analogous to MFD. This leads to recruitment of THIIH and many of the same factors required for GGR.

excision; BER; NER; MMR

_________repair = remove something and replace it; always require a template to replace the thing you removed

____ = remove base (minor base damage)

____ = remove nucleotides from phosphodiester (bulky lesions)

____ = removes mis-incorporated bases

non-homologous end joining; homologous recombination

Double-strand breaks (DSB) Inter-strand crosslinks

_________(NHEJ) __________(HR)

Murine  knockouts in many factors develop tumors with 100% penetrance or embryonic lethal BRCA1 and BRCA2 alleles associated with breast, ovarian, prostate and pancreatic tumors Many rare hereditary cancer predisposition syndromes.

o6 methylguanine; alkylating; guanine

Direct Reversal of Guanine Methylation by O6-meG-DNA-methyltransferase:

__________ nucleotide: Mutagenic because it can ambiguously base pair with either C or T during replication

Mgnt expression status of human tumors is relevant with regard to their sensitivity to________chemo agents

________nucleotide: base pairs only with C

pyrimidine dimers; placental mammals

Direct Reversal of UV Damage by E. coli photolyase:

•Photolyase systems use the energy from light in the visual range to directly reverse_________.

•These proteins are present in all animals EXCEPT________ which have evolved a different system devoted specifically to dealing with thymine dimers

N glycosidic bond; apurinic; apyrimidinic

Base Damage: Base Excision Repair:

DNA glycosidases hydrolyze the__________of their corresponding altered base (red) to yield an_____or__________(AP) site.

glycosylase; lyase

BER: Lyases Open the Sugar Ring

•All BER systems will include a specific________.

•Some will also include a______that opens the sugar prior to its excision.

endonuclease; DSB

After Base Excision:

AP site —> enzyme called AP________—> remove small amount of DNA, gap filled in

SS gap —> if DNA pol ran in at this step —> then would have______ —> if play cards right, end up with rest of steps of pathway

aberrant

Different Glycosylases Recognize Different__________Bases

MUT; hydrolyze

Multiple Bacterial Pathways for Correcting 8-OxoG:

1) Replication can repair

2) Second attempt to repair

Enzyme_____that can recognize 8-OxoG in nucleotide pool —>_______so never incorporated in DNA

If cell has undergone DNA replication —> MutY recognize 8-OxoG and remove A

phosphodiester bond

Bulky Adducts: Nucleotide Excision Repair

DNA endonucleases (or excinucleases) hydrolyze the________ between deoxyribose sugars to yield a nick in the DNA

NER v. BER = type of damage that they address —> bulky lesions block polymerase —> deform the DNA overall deformation of DNA that is recognized —> Block RNA polymerase and recruit NER machinery

linker

Global NER: anywhere in the genome/or can be coupled to RNA polymerase —> Stalls when it comes to bulky lesion —> recruit_______molecule and repair machinery

Cells preferentially repair DNA in region they need to use at that moment