Lec 63 Biochem: DNA III

Mismatch repair pathway increases overall fidelity by polymerization by

1000-fold

What percent of BRCA2 mutation carriers will develop breast cancer?

45% by the age of 70

Deamination of 5-methyl cytosine

5-methyl cytosine deaminates to thymine, which is a standard base and not recognized as a mistake. So, half of the time it will template an G/C to A/T mutation

What percent of BRCA1 mutation carriers will develop breast cancer?

55-65% by the age of 70

result of homologous recombination

A crossover event (DNA flanking the event is exchanged for the length of the molecule) A non-crossover event (DNA exchange is limited to the region)... Depending on how the Holiday junction is resolved

Werner syndrome

A rare disorder characterized by premature aging. People affected by Werner syndrome develop normally until adolescence, at which time they begin to age rapidly. By age 40, they look several decades older. WRN is the defective gene

Nucleotide excision repair

A repair system that removes and then correctly replaces a damaged segment of DNA (e.g. a UV-induced TT dimer) using the undamaged strand as a guide

DNA damage response (DDR) steps

ATM and ATR are the first line sensors that detect damage BRCA1 is involved and p53 is a key target Activation pf p53 blocks cycling, over-activation triggers apoptosis to prevent progression to cancer cell

H3K4me3

Active histone marker associated with the CpG islands that prevents DNA methylation

Deamination of adenine

Adenine can be deaminated, forming hypoxanthine, which pairs with cytosine instead of thymine. This is an AT to GC mutation after replication

BRCA1 role in homologous recombination

BRCA1 activates bound RAD51 to promote invasion

BRCA2 role in homologous recombination

BRCA2 assists RAD51 loading to the resented 3' strands

Translesion synthesis in mammals pathways

Blockage of the fork activates Rad proteins which mono-ubiquitinate PCNA (U). This increases the affinity of PCNA for polη and enables polη to be switched in and to carry out TLS past the damage. After the damage has been by-passed, NOT repaired, replication by polδ restarts

Defects in BRCA1

Breast and ovarian cancer Repair by homologs recombination affected

Defects in BRCA2

Breast, ovarian and prostate cancer Repair by homologous recombination affected

Bloom Syndrome

Cancer at several sites, stunted growth and genome instability DNA helicase needed for recombination affected

Cells survival after UV exposure in white light v dark

Cells survive better after UV exposure if they are exposed to white light than if they are kept in the dark

What are cancer-related large scale alterations in DNA?

Chromosome-specific duplications and gross rearrangements in cancer cells which points to the importance of regulation replication and recombination

CpG islands during tumorigenesis

CpG islands become methylated and is associated with inactive chromatin markers (e.g. H3K9me3) and the downstream gene is sielenced.

CpG islands

CpG-rich sequences of DNA that are usually unmethylated in normal tissues and associated with the active histone mark H3K4me3 which prevents DNA methylation. Promoters are often embedded within CpG islands.

Deamination of cytosine

Cytosine can spontaneously deaminate to form uracil. The presence of thymidine instead of uracil in DNA allows the detection of deamination of cytosine as a uracil that does not belong.

Steps of base excision repair

DNA glycosylase recognizes a damaged based and cleaves the base and deoxyribose An endonuclease (AP endonuclease) cleaves the phosphodiester backbone near the mutation site DNA polymerase I initiates repair synthesis from the free 3' OH at the nick, removing and replacing a portion of the damaged strand (via 5' to 3' exonuclease activity) The remaining nick is sealed by DNA ligases

What is direct repair?

DNA repair in which modified bases are changed back into their original structures. I.e. repair without replacement, no strand cleavage or repair polymerizations. E.g. photoreactivation, dealkylation

What is base excision repair?

DNA repair pathway that first excises modified (damaged or mismatched) bases and then replaces the entire nucleotide

Deamination of bases

Deamination can be mutagenic. Adenine and cytosine can be deaminated

Diseases associated with defects in nucleotide excision repair (NER)

Defects in pathway cause UV sensitivity, xeroderma pigmentosum; some mutations in specific XP genes are associated with increased incidence of tumors and accelerated neurodegeneration and aging.

Photoreactivation repair pathway

Direct repair pathway in which highly conserved photolyase enzyme complexes absorb light photons and transfer electrons to thymine dimers to cleave the cyclobutane ring. This pathway was inferred from observations that cells survive better after UV exposure if they are exposed to white light than if they are kept in the dark.

Dealklyation repair pathway

Direct repair pathway in which the inducible O6-MGT enzymatically removes the methyl group added to G by environmental mutagens. Similar enzymes act on other bases and on other alkyl adducts.

DNApk (DNA protein kinase)

Enzyme that is involved in non-homologous end joining pathway that phosphorylates p53 and other targets to stall replication to allow time for repair. Also activates Artemis.

DNA glycosylases

Enzymes that recognize a damaged base and cleave the base and deoxyribose. There is an army of DNA glycosylases that each favor specific types of base damage or mismatch. E.g. UNG removes uracil Some enzymes remove one base while others replace a stretch of bases

Epigenetic affect on DNA

Epigenetic inactivation of repair gene promoters are carcinogenic. Epigenetic silencing of many repair genes is associated with specific disease.

WRN gene

Gene that encodes a RecQ family helical which probably acts to resolve aberrant chromosome structures that results form mishaps during replication or repair (not the fork helicase). It plays a role in several repair pathways. Defective gene in Werner syndrome

Proto-oncogenes

Genes prompt cell survival or proliferation. Mutation results in a gain-of-function (altered function) that allows unregulated cell proliferation and survival. It can become activator-independent aka an oncogene.

Radiation affect on DNA

High-energy electromagneticradiation, such as x-rays, may cause breaks in the DNA strands.

What are two classes of recombination?

Homologous (homologous DNA sequences) Site-specific (short, specific DNA sequences recognized by recombinases)

What happens when uracil is detected in DNA?

It is removed by uracil DNA glycosylase and the resulting apurinic site (AP) is repaired with the insertion of cytosine

Steps of non-homologous end joining pathway

Ku heterodimer protein binds to broken ends and DNApk and Artemis (nuclease) are recruited DNA protein kinase (DNApk) is activated on binding, phosphorylates p53 and other targets to stall replication to allow time for repair. Active throughout the life cycle. Artemis is activated by DNApk. Exonuclease trim. Ends rejoined by ligase IV

What types of mutations are more probable?

Loss of function mutations are more probable and hence more frequent than gain of function mutations

Diseases associated with defects in mismatch repair pathway

Lynch syndrome (hereditary non-polyposis colorectal cancer or HNPCC)

Lynch syndrome (HNPCC)

Mismatch repair pathway deficiency associated with this syndrome because a signal transduction receptor with a hot spot for mutation (trinucleotide repeat) renders the receptor 24 activator independent

Steps of mismatch repair system

MutS recognizes the mismatch by its distortion to the helix MutL binds MutS and DNA polymerase, then exoI, to direct it to degrade nascent daughter strand DNA, still containing nicks, from its 3' end to the site of mismatch. Instead, E. coli MutH binds the daughter strand on its unmethylated GATC sites, before dam methylase has time to methylate it. Eukaryotes have whole gene families of these Mut genes. DNA polymerase, on site, replaces new strand (the one with the replication induced mutation)

Mutations in class I and II proteins (growth factors and GF receptors)

Mutations changing the structure or expression of these proteins generally give rise to dominantly active oncogenes.

Mutations in class III proteins (signal-transduction proteins)

Mutations changing the structure or expression of these proteins generally give rise to dominantly active oncogenes.

Mutations in class IV proteins (transcription factors)

Mutations changing the structure or expression of these proteins generally give rise to dominantly active oncogenes.

Mutations in class V proteins (pro- or anti-apoptotic proteins)

Mutations changing the structure or expression of these proteins generally give rise to dominantly active oncogenes.

Mutations in class VII proteins (DNA repair proteins)

Mutations greatly increase the probability of mutations in the other classes of proteins.

Nucleotide excision repair (NER) in E.coli to man

NER or "short-patch" repair is conserved from E.coli (uvr proteins) to man (XP proteins)

What enzyme usually modifies mutagens?

P450 enzymes in the liver (mutagens may not be effective unless modified, usually by this enzyme)

PARP (poly ADP-ribose polymerase)

PARP generates signal at nicks to recruit repair proteins to nicks. I.e. it repairs ssDNA nicks.

PARP inhibitors

PARP inhibitors also inhibit ssDNA break repair in non-cancerous cells, but these cells can repair the dsDNA breaks by homologous recombination. BRAC1 defective cancer cells cannot. PARP inhibitors accumulate nicks, which become dsDNA breaks upon replication.

Why are PARP inhibitors effective on cancers with BRCA 1/2 mutations?

PARP repairs ssDNA nicks. PARP inhibitors accumulate nicks, which become dsDNA breaks upon replication. These cannot be repaired effectively in cancers with BRCA1/2 mutations, hence PARP inhibitors are effective on these cancer types

What is mismatch repair system?

Pathway that removes replication errors that escape from the replisome This is responsible for monitoring trinucleotide repeat expansions

Non-homolgous end joining (NHEJ)

Pathway to repair dsDNA breaks that involves re-joining of ends via the Ku protein

Repair pathways that target the mutant strand v. those that do not

Pathways that target the mutant strand are relatively error-free and those that do not are error prone

Defects in MSH2, 3, 6, MLH1, PMS2

Phenotype colon cancer, mismatch repair affected

Defects in Ataxia telangiectasia (AT)

Phenotype leukemia, lymphoma, γ-ray sensitivity, genome instability ATM protein affected

Defects in Xeroderma pigmentosum (XP) groups A-G

Phenotype skin cancer, UV sensitivity, neurological abnormalities Nucleotide excision repair affected

ssDNA break repair pathway steps

Poly adenosyl ribosylase (PARP) recognizes and binds ssDNA breaks PARP binding activates its auto-ribosylation which recruits repair complex of polymerase and ligase PARP disengages, cleaves its polyribose tail, recycles

Branch migration

Process by which base pairs on homologous DNAstrands are consecutively exchanged at a Holliday junction, moving the branch point up or down the DNA sequence Results in an non-crossover event

RecA (eukaryotic Rad51)

Protein involved in homologous recombination. The DNA-protein complex "probes" duplex DNA looking for basepairing. If it finds none, it withdraws leaving the duplex intact. If it finds homology, it displaces one strand and primes DNA polymerization on its newly found template. Protein drives strand invasion

Ku protein

Protein involved in non-homologous end joining and involved in rejoining of ends formed during rearrangement of immunoglobulin genes during B and T cell development. In the later, the ends are formed by site-specific endonuclease activity (not damage).

TFIIH protein

Protein involved in nucleotide excision repair. TFIIH is part of RNA polymerase II and can flag damage for XP protein repair during transcription, so actively transcribed regions of genome tend to be repaired more rapidly

ATM protein

Protein kinase activated by double-strand breaks

Virus encoded proteins

Proteins can activate growth factor receptors and induce cancer

XP proteins

Proteins involved in nucleotide excision repair. Some proteins recognize damage, complex builds on damaged strand only, XP cleavage and removal for replacement synthesis

Hydroxyl radicals affect on DNA

Radicals can oxidize guanine to 8-oxoguanine, which base pairs with adenine instead of cytosine during the next round of replication. This is an GC to AT mutation after replication.

Rec A (Rad51) in homologous recombination steps

RecA-ATP binds cooperatively to the 3' ssDNA to form a distorted nucleofilament The complex explores dsDNA, and is able to distort the helix enough to test whether there is extensive complementarity to is ssDNA cargo IF there is, a conformational change activates ATPase, and recA dissociates, leaving the invading strand and a new partner in the duplex, displacing its identical sequence as a looped-out structure.

Ames test steps

Recover liver from stimulated mouse purifying a fraction (S9) S9, with or without potential mutagen X, is incubated with bacteria that are His- (require histidine because of a single mutation) Bacteria are plated on media without His Number of colonies reflects ability of mutagen from liver to mutate his- to his+

What is ssDNA break repair?

Repair pathway by PARP (poly adenosyl ribosylase) Pathway is important because if it is absent, a replication fork may pass through to convert the ssDNA break into a dsDNA break

Repair mechanisms throughout evolution

Repair pathways are conserved from E. coli to man, usually with increasing complexities. All repair mechanisms survive evolutionary pressure because they are essential.

Affect of triplet repeated sequences

Repeats like CAG tend to be transiently released and picky up again, slightly out of register, by the polymerase enzyme This process is enhanced by the ability of specific trinucleotide repeats to form secondary structures that "pull" the already loose nascent strand from the active site This results in the expansion or contraction of the number of triplet repeats at some loci in the human genome

Bloom's helicase

Resolves crossovers in homologous recombination without recombination Deficiencies result in multiple crossovers, dwarfism, genome instability, predisposition to cancers

Consequences of homologous DNA recombination

Restoration of replication at broken/stalled forks Repair double-stranded breaks in DNA Rearrange genome, generate new combinations of alleles Gene conversion Integration of one genome or DNA into another

Homologs recombination (HR) or homologs end-joining roles in cells

Roles in all cells in rescue of stalled replication and in dsDNA break repair, involves using sister chromatid (identical in sequence) during replication as a template to replace defective chromatid The process is also used in meiosis, where the dsDNA break is induced by spo11 endonuclease, to drive sexual recombination

CHEF (contoured clamp homogenous electrophoresis field) gels

Separate whole yeast chromosomes up to 15 Mbp or very large fragments of human chromosomes, made by digesting with infrequently cutting restriction enzymes like NotI.

What is the Ames test?

Test that measures the effectiveness of chemicals as mutagens after processing in mouse host (liver)

Translesion synthesis

The cell induces alternative polymerases that can replicate across various types of damaged sites. These induced polymerases are often error prone and insert incorrect bases across the damaged sites.

What allows for repair of deaminated cytosine?

The presence of thymine instead of uracil in DNA. If uracil naturally occurred as a base in DNA, cytosine deamination would lead an A-U pair replacing a G-C pair after the next round of replication. And there would be no recognized the U resulting from deamination of C, from a naturally occurring U in this hypothetical scenario.

Rates of mutations as one ages

There is an exponential increase in invasive cancer with age. This means that early mutations to repair pathways increase mutation rates in subsequent generations (early mutations make later mutations more likely). Multiple mutations is needed to convert a normal cell to a cancer cell (cancer is a mutli-hit disease aka accumulated mutations)

DNA repair in cancer cells

These cells have high demand for DNA repair due to their genome instabilities. One chemotherapeutic strategy is to inhibit repair to semi-selectively induce death.

Liver cytochrome P450 enzymes

These enzymes alter toxins to inactive or enable their excretion. This enzyme can lead to modifications that are unintended consequences of exposure of compounds not normally encountered. For instance, aflatoxins an be converted to an reactive epoxide This enzyme can modify environmental agents and mutagens

Mutations in class IV proteins (cell cycle control proteins)

These proteins mainly act as tumor suppressors. Mutations in the genes encoding these proteins act recessively to release cells from control and surveillance.

Aflatoxin B1

This can be converted into a highly reactive epoxide (active DNA-modifying agent) by cytochrome P450. Epoxide reacts with guanine forming a compound that, during replication, converts a G-C base pair into an A-T base pair

UV radiation affect on DNA

UV radiation covalently links adjacent pyramiding thereby blocking replication

Diseases associated with defects in photoreactivation pathway

Xeroderma and pigmentosum

Holiday junction

a branched nucleic acid structure that contains four double-stranded arms joined that forms during homologous recombination

Apurinic site

a site in DNA that is missing a purine base

What happens if the number of trinucleotide repeats surpasses a threshold level?

affected genes are not expressed normally or contain runs of abnormal amino acids, resulting in diseases the number of repeats required, and of course the disease, depends on the locus and the affected gene

What can be converted to reactive epoxide?

chemicals in cigarette smoke, automobile exhaust, and aflatoxin

Mutagens

chemicals that alter specific bases after replication is complete

Diseases associated with defects in dealkylation pathway

colorectal cancer, lung cancer, lymphoma ans glioblastoma

Homologous recombination steps

dsDNA break is recognized and trimmed back with a 5' exonuclease The resulting 3' ssDNA ends become "invasive" with the help of recA (Rad51) The recombination can result in either a crossover event (DNA flanking the event is exchanged for the length of the molecule) or a non crossover event (DNA exchange is limited to the region), depending on how the Holiday junction is resolved

dsDNA breaks

dsDNA breaks are catastrophic lesions in eukaryotes with large genomes (large targets) for ionizing radiation. These breaks are frequently "jagged" and not blunt. Also, induced by stalled replication forks. Breaks are intolerable since they generate chromosomes with no centromeres and sometimes no origins...lethal events. Even in E coli, a single unrepaired ds break is lethal.

What induces translesion and SOS pathways?

extensive damage

Pathways for repairing dsDNA breaks

homologous recombination and non homologous end joining

Defects in Ku, DNA-PK and Artemis result in

immunodeficiences

Why is BRCA1/BRCA2 a tumor suppressor gene?

it is critical in a checkpoint response to dsDNA breaks

COMET assays

lyse cells on agarose, apply current, only broken DNA is small enough to migrate out of original spot

dsDNA breaks are demonstrated in

mitotic chromosomes (microscopy), karyotype, COMET assays, or by CHEF gels

Many environmental agents are carcinogenic only after

modification by the liver cytochrome P450 enzymes

Mutational or epigenetic inactivation of ___ repair pathway(s) causes diseases

one

What can damage bases?

oxidizing agents (hydroxyl radicals), alkylating agents, and light bases can react with hydrocarbons in an alkylating reaction

Cell cycle checkpoint pathways

pathways delay cell cycle to repair and induce repair pathways or apoptosis

Homologous recombination between direct repeat sequences

results in deletion (and insertion) of sequences between the inverted repeats

Homologous recombination between indirect repeat sequences

results in inversion of sequences between the inverted repeats

what would happen without DNA repair?

spontaneous DNA damage would rapidly accumulate and inactive genomes

What causes ssDNA breaks?

ssDNA breaks are caused by ionizing or UV irradiation, some mutagens, ROS, mistakes during BER or mistakes by topoisomerase I

Diseases associated with defects in base excision repair (BER)

subtle disease associations, including cancer, and may have to- be-discovered links to mitochondrial deficiencies and aging, neurodegeneration

What is homologous DNA recombination?

the physical exchange of DNA strands or copying of one template to correct or convert another

UTR

untranslated region of mRNA that can be 5' (upstream) or 3' (downstream) of the ORF (open reading frame)