genetics ch 15

what are the differences between NHEJ and HR

-NHEJ joins DNA ends independently of sequence complementarity while HR uses sequence complementarity on a homologous chromosome to extend DNA past the break -NHEj is more prone to incorporating errors while HR is less prone -NHEJ functions in both dividing and non dividing cells while HR functions primarily in S (DNA replication) and G2 phases of the cell cycle

give an example of a Bulky adduct

Aflatoxin B when bound to DNA, Aflatoxin B is called a DNA adduct

what is a DSB

Double Strand Break both strands of a double helix break at sites close together can cause chromosomal aberrations can be spontaneous or induced

what is a point mutation

a single bp change in the DNA sequence

how do mutations arise

from DNA damage (lesions) physical/chemical abnormality of structure

what are the DSBR and SPSA pathways

DSBR - Double Strand Break Repair SPSA - Synthesis Dependent Strand Annealing

what are EMS and MNNG

Ethylmethanesulfonate (EMS) Methylnitronitrosoguanidine (MNNG) alkylating agents which add alkyl groups to positions on bases

what is the Ras protein when is it activated/inactivated

a GTPase that has active/inactive states when bound to GTP/GDP activated by GEFs which exchange GDP for GTP inactivated by GAPs which converts GTP to GDP

what does the G12V mutation do

blocks GTPase activity, locking it in its active form promotes cell proliferations which is uncontrolled with the mutation leads to cancer usually

what is the Ames test, 1970s

way to test if a compound is mutagenic by exposing special mutant bacterial strains to the product formed by that compounds digestion by liver extract and counting the number of colonies. only new mutations presumably produced by the compound can produce revertants to wildtype able to form colonies if lots of colonies are produced, the cmpd is mutagenic bc it added a mutation which allowed it to grow by reverting initial mutation

what are base analogs

when a chemical cmpd that is sufficiently similar to the normal base of DNA is incorporated into the DNA ex. 2-AP (2- aminopurine) is an analog of adenine that base pairs with thymine and mispairs with cytosine when pronated causes transition mutations

what is replication slippage

when loops in single stranded regions of DNA are stabilized by the "slipping mispairing" of repeated sequence in the course of DNA replication hotspots for indel mutations

explain the TLS synthesis pathway in both bacteria and eukaryotes

1. initiated by stalled DNA pol --> recruits translesion (TLS) pol --> synthesis past lesion - in E coli, this is Pol III - in euks, this is pol epsilon they stall at depurination, bulky adduct, and pyrimidine dimer sites - TLS pol is recruited by replicative beta clamp in E coli and PCNA in eukaryotes - in E coli, there are three TLS pols (Pol II Pol IV and Pol V) - in euks, there are 5 pols (Pol [fancy n] Pol [funky greek letter idk] Pol [fancy k] Rev1, and Pol[funky Z?]) in E coli, TLS is activated by SOS response --> emergency response to prevent cell death

what are the two types of direct DNA damage repair

1. photoreactivation 2. base alkylation reversion

what are the 2 types of mutational inhertitance in multicellular eukaryotes

1. somatic mutations - those which arise in single cells such as human skin or liver cells during an organisms life and are passed onto daughter cells but are not inherited by offspring 2. germline mutations - those in gametes such as human sperm/egg which are inherited by offspring and are present in all of their cells

what are missense (nonsynonymous) mutations

change in the sequence of a codon to one that codes for a different amino acid

what are three differences between replicative polymerases and TLS polymerases

-TLS pols have a larger active site so that they can accomodate damage while rep pols stall bc damage does not fit into active site - TLS pols are error prone bc they done have 3'->5' proofreading activity that rep pols have -TLS pols have low processivity while rep pols have high processivity

what are the steps of SDSA

1. DNA ends processed by exonucleases to generate 3' single strand overhangs 2. enzyme recombinases allow invading 3' overhang to displace one strand of homologous DNA and base pairs to the other -this forms displacement loop (d-loop) 3. invading strand is extended by DNA synthesis using homologous strand as template (^^SAME AS DSBR) 4. DNA helicases displace extended invading strand 5. annealing of OG broken chromosome pieces 6. DNA synthesis 7. DNA ligation

what are the steps of DSBR

1. DNA ends processed by exonucleases to generate 3' single strand overhangs 2. enzyme recombinases allow invading 3' overhang to displace one strand of homologous DNA and base pairs to the other -this forms displacement loop (d-loop) 3. invading strand is extended by DNA synthesis using homologous strand as template (^^SAME AS SDSA) 4. other 3' overhang invades forming a 4-branched, double crossover intermediate- a double Holiday Junction (HJ) 5. HJ resolution - endonucleases calld resolvases cleave the HJ's to yield either crossover or noncrossover segments 6. gaps filled by DNA pols 7. DNA ligase seals nicks

give examples of how point mutations in DNA/RNA reg elements can affect molecular processes

1. PMs in eukaryotic pre-mRNA splice sites block complementary base pairing of small nuclear RNAs which will affect removal of introns 2. PMs in eukaryotic mRNA UTRs block binding of proteins or microRNAs will affect TL 3. PMs that block intramolecular base pairing affect the function of RNAs 4. PMs can create new regulatory elements such as enhancers that effect TC and splice sites affecting RNA processing

what are the two kinds of pyrimidine dimers that UV light can form in DNA how do they cause mutations

1. cyclobutane pyrimidine dimers --> characterized by a cyclobutane ring with Carbons 5 and 6 of adjacent pyrimidines 2. 6-4 photoproducts --> have linked carbons 6 and 4 of adjacent pyrimidines these dimers stall DNA pol bc the bases cannot specify a complementary partner through H bonding

explain BER steps in eukaryotes

1. damage detected by DNA glycosylase which cleaves the glycosidic bond, creating AP site 2. AP endonuclease nicks the strand upstream of AP site (***first two steps same in bacteria and euks) 3. DNA Pol beta inserts a nucleotide before excising AP residue (short patch BER) **in long patch BER, Pol beta inserts 2-10 bases, preventing excision of AP residue 4. Pol beta creates a flap, a single stranded DNA region, which is removed by Flap endonuclease 5. DNA ligase heals both branches

explain BER steps in bacteria

1. damage is detected by DNA glycosylase which cleaves the glycosidic bond, creating AP site 2. AP endonuclease nicks the strand upstream of AP site, DNA phosphodiesterase removes the AP sugar-phosphate 3. DNA pol fills in gaps, DNA ligase heals nick

list the steps of mismatch repair in eukaryotes

1. detection by MutS like complex of Msh2 and Msh6 proteins which interact with a MutL like complex of Mlh1 and Pms1 proteins which incise newly synthesized strand - incision is activated by DNA replication beta clamp PCNA 2. error is removed, 2 DNA pols synthesize and DNA ligase seals

list the steps of mismatch repair in bacteria

1. detection by MutS protien, binds and distorts DNA and recruits MutL and MutH 2. MutH cuts new strand with incorrect base -MutS and MutL also activate excision involving DNA helicase UvrD and 4 strand exonucleases. 3. UvrD unwinds DNA toward mismatch 4. DNA pol III repairs gap, DNA ligase seals

list steps of NHEJ

1. detection by a heterodimer of Ku70-Ku80 that binds to each broken end this promotes further damage and recruits DNA-Pkcs 2. DNA-Pkcs (DNA protein kinase, catalytic subunit) phosphorylates the nuclease Artemis, activating endonuclease and 5' exonuclease activities this removes the 5' and 3' DNA overhangs/hairpins 3. DNA pol mew and DNA pol lambda fills in gaps this leaves 5' phosphate and 3' hydroxyl ends open 4. XRCC4 XLF (XRCC4 like factor) complex and DNA ligase IV seals gap NHEJ does not join telomeres

list the steps of NER in bacteria

1. detection by either UvrA/UvrB (GG-NER) or by RNA pol which stalls during TC at damage and brings UvrA-UvrB (TC-NER) 2. UvrA-UvrB has helicase activity and separates strand 3. UvrA leaves, UvrB is a scaffold for UvrC which uses endonuclease domains to cleave phosphodiester bonds 8 nucleotides 5' and 4-5 nucleotides 3' of damage 4. DNA helicase UvrD and DNA Pol 1 excise the 12-13 nucleotide region between incision sites and fill in gaps 5. DNA ligase seals nick

list the steps of NER in eukaryotes

1. detection by either XPC and XPE (GG-NER) or RNA Pol II, which stalls during TC at damage and brings CSA and CSB (TC-NER) 2. Both XPC-XPE and CSA-CSB recruit TFIIH complex which also functions as RNA Pol II in TC 3. 2 subunits of TFIIH, XPB and XPD have helicase activity which seperates DNA strands 4. XPA and RPA expand DNA bubble around damage 5. XPF-ERCC1 endonuclease cleaves a phosphodiester bond 5' of damage and TFIIH endonuclease XPG cleaves 3' of damage (27 nts) 6. gap is filled by DNA replication factors (RPC (replication factor C), PCNA (proliferating cell nuclear antigen) and 2 kinds of DNA pols) 7. DNA ligase III-XRCC1 (xray repair cross complementing protein 1) or DNA ligase 1-FEN1 (Flap endonuclease 1) seals nick

how is mismatching usually detected what happens if they arent detected

3' --> 5' exonuclease activity of DNA pol if not detected, they become transition mutations

what is an example of a trinucleotide repeat disease

Fragile X Syndrome results from an increase in the number of CCG repeats in the 5' UTR of the FMR1 (fragile x mental retardation) gene silencing of FMR1 gene by DNA methylation of CpGs in an island at the FMR1 promotor surrounding regulatory regions and in the CCG repeats results in full mutated allele with more than 200 repeats

what are GEFs

GTP exchange factors exchange GDP for GTP in the GTPase Ras protein

what are GAPs

GTPase activating proteins intrinsic GTPase activity converts GTP to GDP to inactivate Ras protein

what are ROS

Reactive Oxygen Species ex. superoxide radicals (*O2-), hydrogen peroxide (H2O2), and hydroxyl radicals (*OH) are byproducts of normal aerobic metabolism of molecular oxygen by mitochondria

what is the difference between TC-NER and GG-NER

TC-NER - transcription coupled - machinery comes to problem when TC stalls GG-NER - global genome - machinery constantly scanning DNA for issues

how does UV light induce mutations

UV light at about 300 nm forms various types of pyrimidine dimers in DNA

what was the correlation that the Ames test found/ what was the result

a strong correlation between the ability of chemical compounds to cause cancer and the ability to cause mutations not all compounds are directly mutagenic, the actual mutagenic agent is sometimes a metabolite of the compound that is produced in the body (typically enzymes in the liver)

what is alkylation

addition of an alkyl group (CnHn+1) ex. a methyl group (CH3) or an ethyl group (C2H5) to a base done through alkylating agents (EMS and MNNG) which add alkyl groups to many positions on all 4 bases

what are the four mechanisms of induced mutations (from exogeneous chemical agents)

alkylation, bulky adducts, base analogs, intercalating agents

what are the three types of point mutations & give def

base substitutions - one bp replaced with another (transition or transversion base insertions - addition of one base pair base deletions - removal of one base pair (insertions/deletions called indel mutations)

what are bulky adducts

causes mutations by attaching to guanine at the N-7 position and leads to breakage of glycosidic bond, creating AP site

what are frameshift mutations

change in the TL reading frame for all codons downstream of a mutation which is caused by an indel mutation

what are synonymous (silent) mutations

change in the sequence of a codon but not the encoded amino acid

what are nonsense mutations

change in the sequence of a codon that codes for an amino acid into one that stops translation

what are mutations :I

changes in DNA sequences that cannot be repaired that are transmitted through DNA replication

what are the two types of missense mutations & give def

conservative - amino acid is replaced with a different chemically similar one (may not significantly after the proteins structure and function) ex. a change in size or polarity nonconservative - amino acid is replaced with a chemically different one which is likely to produce a change in the protein's structure or function

explain photoreactivation

cyclobutane pyrimidine dimers (CPDs) caused by UV light are repaired by CPD photolyase enzyme it binds to CPD, breaks covalent bond, and regenerates old bases requires light for energy (mammals done have this)

explain the 5 basic steps of the NER pathway

damage detection, strand separation, incision, excision, polymerization, ligation

what do mutational hotspots correspond with in the lacI gene

deaminations at cytosine residues in lacI gene 5-methylcytosine residues are present the deamination of this generates thymine which is not recognized by uracil-DNA glycosylase and is not repaired

what are the three mechanisms of spontaneous mutations

depurination, deamination, and oxidative damage

what are trinucleotide repeat diseases

diseases caused by a mutation where the number of 3-nucleotide repeats is increased over a stable threshold

what does the human ras gene do what does a mutation in the gene do & effect of this

encodes the Ras protein changes a glycine at position 12 to a valine (G12V) glycine has a conformational flexibility that other amino acids dont have --> changing the amino acid can have severe effects on protein function

what is Ionizing Radiation (IR) and what does it create

from gamma rays and Xrays cause mutations by generating ROS through radiolysis of water and disruption of mitochondrial functions leads to generation of thymine glycol and 8 - oxoguanine, leading to mutations can also directly damage DNA, leading to AP sites (Abasic sites) bc it severs phosphodiester backbone

what is HR

homologous recombination requires an undamaged homologous double stranded DNA template

what did the Luria and Delbrück fluctuation test, 1943 figure out

hypothesis - if mutations were spontaneous, they would occur at different times in different E. coli cultures - earlier on mutations would create a higher # of resistant cells to a virus bc mutant cells had more time to produce more resistant descendents - therefore, if mutations are spontaneous, the # of resistant colonies/cultures would show high variation (fluctuation) Results: the reigning paradigm of mutation = mutations can occur in any cell at any time randomly

what are tautomers

isomers that differ in the positions of their atoms and in the bonds between the atoms (in equilibrium)

what is depurination

loss of a purine base (G or A) caused by hydrolysis of the glycosidic bond between the base and deoxyribose sugar loss of base,, phosphodiester backbone intact

are mutations always bad

no they can be beneficial, detrimental, or neutral

what is NHEJ

non-homologous end joining mechanism which processes and rejoins DSB ends, frequently creating indels at break sites

what are the two primary pathways for DSB repair

nonhomologous endjoinging (NHEJ) or homologous recombination (HR)

what are auxotrophs,, what is an ex

organisms which require nutrients for growth not needed by a wildtype strain Salmonella typhimurium -- can only grow with the amino acid histidine because it has mutant alleles for histidine synthesis the strain can also revert (another mutation can be added, allowing for histidine synthesis)

what are intercalating agents/how to they work

planar molecules that mimic base pairs and are able to slip themselves in (intercalate) between the stacked bases inside the DNA helix ex. Proflavin and acridine orange (agent orange) they distort the DNA duplex by fooling the DNA pol into inserting extra bases or skipping templated bases (indel mutations)

what is ionization

process by which an atom in DNA acquires a pos or neg charge brought on by a proton exchange between water H bonds can allow DNA pol to insert a mismatch that is like a wobble base pair

what is translesion synthesis generally

provides additional time for other mechanisms to repair the lesion before the replicative DNA pol returns to finish synthesizing the genome

what is mismatch repair

reduces error rate in replication by recognizing and repairing mismatched bases and small loops caused by indels of nucleotides

what is Nucleotide Excision repair (NER) generally

relieves replication and transcription blocks by repairing the damaged DNA

what are DNA/RNA regulatory elements

short and serve as binding sites for proteins/RNSa that catalyze or scaffold molecular proceses point mutations can disrupt this process

what would depurination result in without repair systems

significant genetic damage bc Apruinic (AP sites) cannot specify a complementary base

what is oxidative damage

spontaneous lesion that generates mutations thymine --> thymine glycol (no base pairing,, no replication) guanine --> 8-oxoguanine (8-oxoG) (mispairs with A --> G-C to T-A)

how does strand recognition work in mismatch repair in bacteria

strand recognition by MutH is directed by adenine methylation at GATC sequences -occurs after synthesis --> the DNA is temporarily unmodified MutH endonuclease cuts unmethylated strand at hemimethylated GATC sequence (stimulated by MutS and MutL)

what is base excision repair (BER) generally

targets non bulky damage to bases produced by alkylation, oxidation, and deamination

what are the two processes that cause mismatching

tautomerization and ionization

explain the SOS pathway/response

the SOS pathway regulates production of DNA repair and damage tolerance proteins last resort bc its trading cell death for some kind of mutagenesis (doing the wrong thing is better than doing nothing,, increases chances of survival)

give an example of how base alkylation is reversed in direct DNA damage repair

the alkyltransferase O6 - methylguanine DNA methyltransferase (MGMT) repairs O6 methylguanine back to guanine by transfering a methyl group from O6 to a cysteine residue in its active site this permanently inactivates the enzyme, making it a suicide enzyme

what is deamination

the hydrolytic removal of an amine group which alters the 3 DNA bases tha contain an amino group - converts cytosine to uracil, adenine to hypoxanine, and guanine to xanthine deamination of 5- methylcytosine (5mc) produces thymine (C-G to T-A)

what is the difference between mismatch repair in bacteria and eukaryotes

the method of strand discrimination

what is recombination

the outcome of cellular processes that cause alleles in different genes to become grouped in new combinations

what is tautomerization and what does it cause

the spontaneous isomerization of a base from its normal (Keto) form to an alternative hydrogen bonding enol (or imino) form the imino/enol forms may pair with the wrong base, forming a mispair --> mismatching

how many DNA glycosylases do cells have, whats the point

they have many which recognize different types of damaged bases ex. uracil-DNA glycosylase removes uracil from DNA

what is the sim/dif between NER in bacteria/eukaryotes

they have the same steps but there are more enzymes involved with eukaryotes and there is a larger (27 nts) excision area in euks compared to 12-13 nts in bacteria

what are induced mutations

those which arise through the action of external agents, mutagens, that increase the rate at which mutations occur

what are spontaneous mutations

those which occur naturally and arise in all cells

what are the two types of base substitutions & give def how many possible combos of mutations in each

transition - replaces a purine with a purine (A to G or G to A) or a pyrimidine with a pyrimidine (C to T or T to C) transversion - replaces a purine with a pyrimidine (A to C, A to T, G to C, or G to T) or a pyrimidine with a purine (vice versa) 4 possible transitions 8 possible transversions

what are the two mechanisms of induced mutations (from exogenous physical agents)

ultraviolet light and ionizing radiation (IR)

can mutations in nc (noncoding) regions affect phenotype?

yuh