week 6 genetics exam 2

active p53 increases expression of genes that inhibit DNA synthesis prior to the G1/S checkpoint and also inhibit passage the G2/M checkpoint

DNA damage --> activated p53 ??

base excision repair takes care of minor base damage

the base excision repair pathway takes care of non bulky damage to bases including methylation, deamination, oxidation, spontaneous loss of a base

failure to stop at cell cycle checkpoint

(cause) mutation in a gene that usually slows the cell cycle --> (effect) rate of cell division is accelerated (cause) failure to pause for DNA repair --> (effect) faulty DNA leads to unregulated cell growth and genome instability (cause) loss of control over telomere length --> (effect) cancer cells have telomerase, an enzyme that elongates telomeres - cells continue to divide after 50 mitosis

testing for mutagenicity w/ the Ames Test

(strains are used that are his- as a result of either a base substitution or a frameshift mutation) took liver from mouse; grind liver and centrifuge; mix and plate (incubated on plates containing a compound of interest, a liver extract, and enough histidine to sustain a few rounds of replication); his+ revertant colonies are counted -provides an important way of screening compounds and evaluating their risk to our health and to the environment

error prone repair- preventing the occurrence of more serious outcomes

*DNA damage bypass {replication stalls, then reinitiates beyond damage; gap filled by strand exchange w/ undamaged parental strand and subsequent repair synthesis} *Template-direct gap repair {single stranded 3' end of undamaged strand invades the alternate molecule and uses it for a template for synthesis; subsequent release allows that newly synthesized strand to act as a template for gap repair

depurination

the bond b/w sugar and base is broken by reacting with water, base is replaced by -OH and diffuses away

bases can be damaged do that no specific base pairing is possible

-UV photoproducts -ionizing radiation reacts w/ water to form reactive oxygen species including the highly damaging 8-oxo dG and thymidine glycol

driver and passenger mutations

-driver = mutations causing the selective growth advantage (cause the cancerous state) -passenger = majority of mutations are likely to be passengers (don't contribute to the development of cancer but have occurred during the growth of cancer)

template directed gap repair - an error free method

-gapped DNA molecules w/ 3' overhangs --> invasion of the template and elongation of the 3' end --> release of the elongated 3' end --> reassociation of the 3' end with its original partner --> new synthesis across the single stranded gaps restores the original molecule except for possible differences picked up from the template

loss of heterozygosity in familial retinoblastoma

-sparatic disease, common in families -in the individual inherits a mutant RB1 gene; later the wildtype RB1 allele is lost by one of the mechanisms above which uncover the recessive allele (loss of heterozygosity); the germ line mutation itself does not itself cause cancer, but does make it more likely that progression will occur

cell cycle checkpoints

-spindle assembly checkpoint in the end of mitosis -DNA damage checkpoints in G1, S, G2 -Apoptosis checkpoint in the beginning of mitosis

summary of 4 stages of NER process

1) recognition of damaged base 2) assembly of a multiprotein complex at the site 3) cutting the damaged strand several nucleotides upstream and downstream and removal of intervening nucloetides 4) use of the undamaged strand as template for DNA polymerase

nucleotide excision repair (NER) corrects bulky adducts that distort the DNA helix

1) recognition of damaged base {NER pathway is activated when bulky adducts or multiple damages based are recognized in nontranscribed or transcribed regions of the genome}{there are two pathways: GG-NER for situations anywhere in the genome that cause replication fork to stall; TC-NER = repairing transcribed regions of DNA} 2) in the first shared step {in the first shared step, both DNA damage recognition complexes recruit the TFIIH complex to the lesion} (TFIIH = large multi-protein complex that includes two helicases, XPA and XPD, that unwind and open the DNA helix around the lesion) 3) the XPb and XPD helicases unwind the DNA helix around the lesion 4) XPF and XPG excise the damaged DNA segment 5) DNA polymerase fills the gap, using the complementary DNA strand as template 6) the new DNA strand is ligated in place

an oncogene may cause excessive response to a growth factor: HER2 overexpression

25% of breast cancer cases are caused by excess copies or high levels of transcription of the HER2 receptor gene; cancer cells contain 1-2 million copies of the HER2 gene; HER2 protein is a receptor for the EGF growth factor, when EGF binds, the HER2 protein becomes a tyrosine kinase which signals to cell to divide

activation of proto-oncogene

??

a fundamental characteristic of cancer is disruption of the _________________

cell cycle; driver mutations may impact: cell cycle checkpoints, length of telomeres, chemical signals from within and outside the cell

p53 becomes activated when DNA damage is detected

DNA damage includes damage to bases or to backbone or incomplete replication; phosphorylation of p53 in response to DNA damage converts it into an active TR with effects on genes that play a central role in triggering the DNA damage checkpoint -in normal cells, p53 levels are low; Mdm2 removes p53 from the nucleus and leads to its degradation by the proteasome -damage to DNA results in p53 phosphorylation (P) and acetylation (Ac) and activation of p53 as a transcription factor -- Mdm2 cannot bind to modified p53 -- so now p53 escapes pathway that usually keep it in check -activated p53 leads to activation of genes involved in cell cycle arrest and DNA repair pathways

replication slippage

DNA poly strand slippage has been proposed as the primary mechanism for instability of SSR; during replication the repeats can misalign resulting in an extrahelical DNA loop that increases in length if it occurs on the template strand

tumor suppressor gene: p53

DNA transcription factor that is activated in response to DNA damage; activated p53 up-regulated genes that prevent progression of the cell cycle until DNA damage is repaired (DNA damage checkpoint) -- *in the absence of p53 the cell cycle progresses even if damaged DNA has not been repaired* -this means that a p53 deficiency leads to an increase in mutations overall; chromosomal rearrangements and aneuploidy are also increased; the spike in mutations increases the chance that additional mutations will arise that promote cell proliferation or block apoptosis

mismatch repair pathway excises a patch containing the mismatch

E coli DNA is methylated on adenine residues; the old strand is methylated, the new strand will soon be but is not yet, thus its possible to distinguish which strand needs to be corrected; in E coli mutH makes a nick at the nearest GATC on the newly synthesized (unmethylated) strand; exonuclease degrades that strand until just beyond the mismatch; DNA polymerase and ligase repair the gap; Dam methylase methylates the A in the daughter strand (no longer able to distinguish the pair)

tumor suppressor gene: RB

RB controls the G1/S transition by controlling the activity of the E2F TF; loss of RB frees E2F to intitate transcription of DNA synthesis enzymes at all points in the cell cycle - thus DNA synthesis is continuously being initiated

formation of Ras oncoprotein

Ras is inactive = GDP bound Ras is active = GTP bound a Ras oncogene can result from a simple point mutation; mutant form of Ras can not hydrolyze GTP to GDP so it remains GTP-bound even in absence of normal signals; the Ras oncoprotein propagates a signal that promotes cell proliferation (again normally would need growth factor to bind for Ras and GTP to combine, but w/ the mutation GTP is constantly bound)

mutagenic effects of UV light

absorption of light energy causes covalent bonds b/w adjacent thymines or cytocines (cross links b/w) which results in distortion of double helix; these dimers can block DNA replication, DNA poly has trouble reading the dimer - incorrect pairing causes mutation -every second you are in the sun 50 to 100 of these dimers are formed in each skin cell {fortunately we have a powerful repair mechanism that takes care of these}

deamination is problematic in both prokaryotes and eukaryotes bc it cant always be repaired

U can be removed from DNA; it is recognized as abnormal and is removed by uracil glycosylase; but T is not an abnormal base, as a result deamination of 5-methylcytosine leads to GC to AT transition mutations

the SOS system (e coli)

UV light induces the synthesis of the RecA protein; when Pol III stalls at the site of damage, the DNA ahead of the polymerase continues to be unwound and is coated by SSb -- together RecA and SSB form filament; activated filament from RecA induces the expression of other genes including the bypass polymerases; Pol III is replaced by a bypass polymerase that can tolerate large adducts and replicate beyond the lesion; bypass polymerases are error-prone in part bc they lack 3' to 5' exonuclease activity; another property of the bypass polymerase is that it isnt very processive (it falls off easily)

xeroderma pigmentosum and the cockayne syndrome patients have defects in the NER pathway

XP: patients are sensitive to UV light (half of kids w this condition develop their first skin cancer by age 10); mutations in many of the NER genes; UV damage genes that control cell growth and division, cells can either die or grow in an uncontrolled way; unregulated cell growth can lead to the development of cancer Cockayne snydrome: patients are senstive to UV light; neurodegenration and intellectual disability; patients age prematurely and die young; mutations in repair genes ERCC8 and ERCC6; no predipistion to cancer

5-methylcytosine is a hotspot for muation

analysis showed that 5-methyl cytosine residues are present at each hotspot

induced mutations

base analogs, specific mispairing, intercalating agents, base damage

neurons are especially sensitive to repeat expansion

buildup for a toxic product produced by the mutant allele underlies late-onset neurodegenerative diseases caused by repeat expansions; the effect of toxic products is especially prominent in neurons because the are long lived and not replaced when lost

mispairing promoted by base analogs

chemicals that look like the base so they can be added into the DNA ex): 5-bromouracil

mutational steps leading to colon cancer

colon lining cells divide more frequently when the APC gene is deleted or mutated forming adenomas; activation of oncogenes like Ras fuel growth; mutations in p53 and other genes push the adenoma cells to become cancerous

how to distinguish driver from passenger mutations.......

count and compare tumor mutations in tumor cells from people at different stages of the same type of cancer; the older the tumor the more genetic changes will have accumulated (a mutation present in all stages amoung several individual's tumors must be acting early in the disease process whereas a mutation seen only in individuals whose disease is further along occurred late)

cancer is not a heritable disease

each case has its own spectrum of mutations that arise in the DNA of somatic cells; these mutations are NOT transmitted from parents to children -- however cancer predisposition IS heritable

radiation

emmission of energy as electromagnetic waves or as moving subatomic particles, especially high energy particles that cause ionization

cancer is a group of diseases that__________

escape the normal controls on cell division -properties: cancer cells divide more frequently, cancer cells are not inhibited by contact w/ other cells and can form tumors, cancer cells can invade other tissues, a process called metastasis

a variety of different glycosylases are used by the base excision repair pathway

excision of uracil results in a apyrimidinic site (deamination of C creates U; U exicised by DNA uracil glycosylase) excision of 8-oxoguanine results in an apurinic site (oxidation of G created OG; OG exicised by DNA oxoguanine glycolyase)

studies of familial and sporadic retinoblastoma led to the two hits concept

hypothesis was that retinoblastoma requires two mutational events; in the familial form, one hit is inherited in the germline, and the second occurs in somatic cells; whereas in the sporadic form, both hits are somatic in origin

ionizing radiation

includes Xrays, particles and radiation released by radioactive elements; when ionizing radiations interacts w water or tissue, free radicals are formed that react with other molecules including DNA causing carcinogenic and mutagenic effects

mutagenesis by 5-BU

incorporation of Bu in its mutagenic pairing configuration is rare; normal pairing of an already incorporated Bu is frequent; the result is a GC->AT transition OR incorporation of Bu in its nonmutagenic pairing configuration is common -> mispairing of an already incorporated Bu is rare -> the results is a AT to GC transition

direct reversal of alkyl group addition caused by EMS or NG

methyl and other alkyl groups bound to the O6 position of guanine in DNA are removed via a thioester linkage to a cysteine residue in the active site of the human MGMT enzyme; this is accomplished without cleavage of the phosphodiester backbone; alkylated MGMT is then degraded

Nuclear accident at Chernobyl

millions of radiation released --- the radioactive iodine was deposited in pastures eaten by cows who then concentrated it in their milk which was drunk by children

post replication mismatch repair

misincorporated bases are 99% removed by the proofreading function of DNA Polymerase; after replication any remaining mismatched bases are 99.9% removed by the mismatch repair system; only 10^-10 bases remain uncorrected in any given replication cycle

cancer has a stepwise origin

mutation inactivates tumor supressor gene --> cells proliferate (grow) --> mutation inactivates DNA repair gene --> Mutation of proto-oncogene creates oncogene --> mutation inactivates several more tumor supressor genes --> cancer

oncogenes: gain of function mutations promoting cell proliferation

mutations of: Ras & growth factor receptors = continual growth signaling; cyclin D and Cdk 4 = unscheduled entry into S phase; other examples in lect

non-homologous end joining

non-homologous end joining repairs double strand breaks by ligating the free ends back together - here there is no potential to correct the problem using an undamaged strand or sister chromatic; two proteins KU80 and KU70 bind to the broken ends (this prevents further damage and also recruits other proteins that create 5' P and 3' OH ends needs for ligation); the XRCC4 ligase protein stitches the ends back together (this process is error prone but its better than the alternative)

tumor suppressor genes: loss of function promotes cell proliferation

p53 (effects loss of G1/S and G2/M checkpoints) RB (effects promotes proliferation; E2F uninhibited) loss of function mutations in genes that normally negatively control cell proliferation or that activate the apoptotic pathway contribute to cancer progression (typically recessive)

intercalacting agents insert between bases of the DNA double helix

planar molecules like profalvine and acridine orange can slip between stacked bases within the helix; this can causes an insertion or deletion during replication

protooncogenes and oncogenes

proto-oncogenes normally trigger cell division (gas pedal) - they are active where and when high rates of cell division are needed; if they are transcribed and translated too rapidly, too frequently, or at the wrong time in development: they = oncogenes (a proto-oncogene can also become as oncogene by being physically next to a highly transcribed gene) again mutations in these are DOMINANT

double stranded breaks are deleterious but are also a fundamental aspect of meiotic recombination

recombination takes place after the replication fork passes through a region forming two chromatids from each homologous chromosome; one chromatid recombines w a non sister chromatid from the other homolog; during meiosis there must be at least one crossover event per chromosome recombination is initiated when Spo11 enxyme makes a double stranded cut; Spo11 remains attached to the 5' end to protect it and to recruit other proteins needed for recombination; 5' ends are then resected (trimmed back) and a protein complex that includes Rad 51 binds the 3' end; the nucleoprotein then invades in a search for complementarity

cancer progression is due to an initial _________________

somatic or germ-line gatekeeper mutation followed by a series of somatic driver mutations

replication errors and spontaneous lesions generate most spontaneous base subsitutions

spontaneous lesions = depurination (loss of purines = apurinic sites which wld be serious if they persisted bc they cant specify a complementary base); deamination (causes transition); oxidative damage (reactive oxygen molecules can damage DNA)

BCR-ABL fusion protein is caused by fusion of Chr 9 and 22

the abl gene encodes a tyrosine kinase; the N terminus of the protein inhibits the activity of the catalytic domain (has folds over on active site); BCR-ABL fusion protein the Abl-N terminus of Abl is missing, so the fusion creates a constitutively active kinase; the overactive tyrosine kinase overrides normal deactivation of the signal transduction pathway (after the signal has stopped), so it continues sending signals into the cell causing it to divide too many times

bypass repair in eukaryotes

the bypass polymerases are always present (not induced) in eukaryotes, therefore the activity needs to be regulated; the solution is the PCNA protein which serves as a sliding clamp during replication to keep Pol III attached to the DNA -- PCNA is ubiquitinated by Rad6 which changes its conformation and allows PCNA to associate with the bypass polymerase allowing translesion synthesis

direct reversal of damaged DNA

the cyclobutane pyrimidine dimer can be reverted by CPD photolyase; CPD photolyase binds to the dimer and splits it; this process requires light - so its called photoreactivation; can repair 80-90% of the CPD

the gatekeeper gene

the gene in which the initial mutation occurs; mutation in the gatekeeper enables a normal cell to divide just slightly faster than others; a clone of faster dividing cells gradually accumulates; later additional mutations increase the division rate even further in these fast dividing cells increasing their proportion in the tissue

specific mispairing is caused by alkylating agents like EMS

these are not incorporated into the DNA; instead they alter a base by adding alkyl groups to various positions on the base and cause it to mispair

genetic mutations that can cause cancer

three major classes of genes are recognized in cancer genetics: -oncogenes {mutated copy of proto-oncogenes that normally has a role in cell division but now promotes cancer development, usually dominant} -tumor suppressor genes {genes are normally required to supress cell division; these mutations are recessive, mutation/inactivation of both alleles are required} -DNA repair genes {genes that encode proteins that are needed for maintenance of chromosome integrity and repair of damage to DNA sequences

naturally occurring mutagens

water (causes depurination) UV light (causes formation of pyrimidine dimers) Ionizing radiation (single and double stranded breaks in DNA)

double strand break repair

what happens if there is no opportunity for repair based on complementarity aka when both strands break -- if these were left unpaired double strand breaks could cause cell death or create a pre-cancerous state

xrays are mutagenic

xrays can cause single strand breaks, double strand breaks, and alterations in nucleotide bases