Genetics Ch 16: Mutation, Repair, and Recombination

types of point mutations: - Base substitutions: - 2 subtypes of base substitutions:

- one base pair is replaced by another - transitions and transversions

replication slippage

If loop occurs in template strand during replication, DNA polymerase misses looped out nucleotides, and small insertions and deletions occur

Intercalating agents: - intercalating agents form DNA modifiers which includes - proflavin, acridine orange, and a class of chemicals called ICR compounds:

distort DNA during synthesis to induce single nucleotide pair insertions and deletions (frameshift mutations) - proflavin, acridine orange, and a class of chemicals called ICR compounds - planar molecules that mimic base pairs and are able to slip in between the stacked nitrogen bases at the core of DNA double helix → they then cause an insertion or deletion of single nucleotide pair

Mutagenesis:

production of mutations in the lab through exposure to mutagens and the organisms is mutagenized

Indel mutations arise when loops in single-stranded regions are stabilized by the ......

"slipped mispairing" of repeated sequences in the course of replication → called replication slippage

Can the existence of mutants in a population be demonstrated before selection? - Replica plating: (lederberg's experiment) a population of bacteria was plated on a nonselective medium (......) and from each cell a colony grew → this place was called ....... → sterile piece of velvet was pressed down lightly on the surface of master plate and then velvet picked up cells wherever there was a colony → it picked up a colony "imprint" from the whole plate and was then touched to replica plates contain selective medium (containing T1 phage) → cells clinging to the velvet are inoculated onto the replica plates in same relative positions as those of the colonies on the original master plate → what was found on the plates and from the experiment?????

- (medium containing no phage) - the master plate - rare resistant mutant colonies were found but the plates showed identical patterns fo resistant colonies → if the mutations had occurred after exposure to the selective agents, the patterns for each plate would have been as random as the mutations themselves → the mutation events must have occurred before exposure to the selective agent which shows that mutations occur randomly all the time rather than in response to a selective agent

Like nonsense mutations, indel mutations may have consequences on polypeptide sequence that extend beyond the site of mutation → the sequence of mRNA is read by the translation apparatus in register ("in frame"), ..... → the addition or deletion of a single base repair of DNA changes the ...... frameshift mutation:

- 3 bases (one codon) at a time - reading frame for the remainder of the translation process which is a frameshift mutation - cause the entire amino acid sequence translationally downstream of the mutant site to bear no relation to the original amino acid sequence which result usually in complete loss of normal protein structure and function

Postreplication repair: mismatch repair: - Correction by the ..... reduces the error rate to less than 10^-7 - Major pathways that correct the remaining replication errors is mismatch repair: - from these values you can see that mutations leading to the loss of the mismatch-repair pathway can .....

- 3'-5' proofreading function of the replicative polymerase - this pathways reduces error rate to less than 10^9 by recognizing and repairing mismatched bases and small loops caused by insertion and deletion of nucleotides (indels) in course of replication - increase the mutation frequency A LOT and loss of mismatch repair is associated with hereditary forms of colon cancer

DNA glycosylases include uracil-DNA glycosylase that removed uracil from DNA → uracil residues that result from spontaneous deamination of cytosine can lead to a ...... if unrepaired → one advantage of having thymine (5-methyluracil)rather than uracil as the natural pairing of adenine is that ....

- C-to-T transition - spontaneous cytosine deamination events can be recognized as abnormal and then be excised and repaired

Biological Repair Mechanisms: - ..... is the only molecule that organisms repair rather than replace - Most important repair mechanisms is ....

- DNA - the proofreading function of DNA polymerases that replicate DNA as part of the replisome: DNA pol I and DNA pol III are able to excise mismatched bases that have been inserted incorrectly

The molecular consequences of point mutations in a noncoding region: - Noncoding regions contain crucial ...... - At the DNA level the binding sites include sites to which ..... - At the RNA level additional important binding sites include ..... - Functional consequences of any point mutation in parts of a gene other than the polypeptide coding segments are harder to predict and depend on ..... - Mutations that disrupt binding sites can change the ..... - Some Binding-site mutations might completely obliterate a required step in normal gene expression and totally.... - Gene mutations are the change in .... - Many point mutations within noncoding sequences have little/no phenotypic change because ...... → these sites may be functionally irrelevant or other sites within the gene may duplicate their function

- DNA binding sites for points interspersed among sequences that are nonessential to gene expression or gene activity - RNA polymerase and its associated factors bind and sites to which specific transcription-regulating proteins bind - ribosome-binding sites of bacterial mRNAs, the 5' and 3' splice sites for exon joining in eukaryotic mRNAs,and sites that regulatie translation and localize the mRNA to particular areas and compartments within the cell - whether the mutation disrupts (or creates) a binding site - expression pattern of a gene by changing the amount of product expressed at a certain time/tissue or by changing the response to certain environmental cues → these will change the amount of protein product produced but not the structure of the protein - inactivate the gene product or block its formation - DNA sequence of a given gene - they are located between DNA binding sites for regulatory proteins

In the 1st step, UV light induces the synthesis of protein called ..... → when the replicative polymerase (DNA polymerase III) stalls at a site of DNA damage, the DNA ahead of the polymerase continues to be ...... → next RecA proteins join the single-strand-binding proteins and form a protein-DNA filament → RecA filament is the biologically active form of this protein → RecA acts as a signal that leads to ...... → DNA polymerases that can bypass replication stalls have been found in diverse taxa of eukaryotes ranging from yeast to humans → these eukaryotic polymerases contribute to damage-tolerance mechanism called ...... that resembles the SOS bypass system in E.coli

- RecA which is a key factor of DNA repair and recombination - unwound, exposing regions of single-stranded DNA that becomes bound by single-strand-binding proteins - induction of several genes that are now known to encode members of a newly discovered family of DNA polymerases that can bypass the replication block and are distinct from replicative polymerases - translesion DNA synthesis

Error-free repair mechanisms: 1) Direct reversal of damaged DNA: - One case: mutagenic photodimer caused by UV light: cyclobutane pyrimidine dimer (CPD) can be repaired by an enzyme called ....... → photoreactivation occurs bc the enzyme requires ......, other repair pathways are required to remove UV damage in the absence of light of appropriate wavelength (less than 300nm) - Alkyltransferases: enzymes that directly reverse lesions by .......... from E.coli enzyme that transfers the methyl group from O-6 methylguanine to a cysteine residue in the enzymes active site but the transfer inactivates the enzyme so this repair system can be ...... if the level of alkylation is high enough

- Reverse the lesion directly and regenerate the normal base - CPD photolyase which binds to the photodimer and splits it to regenerate the original bases - light to function - removing certain alkyl groups that have been added to position of O-6 guanine by mutagens like nitrosoguanidine and ethylmethanesulfonate, methyltransferase; saturated

Reconstitution of the mismatch-repair system in the test tube in lab of Paul Modrich: conservation of many of the mismatch-repair proteins from bacteria, yeast, humans shows that this pathway is .....

- ancient and important in all living organisms

Incorporation of base analogs: - Some chemical compounds are similar enough to the normal nitrogen bases of DNA that sometimes they are incorporated into DNA in place of normal bases = ........., after in place these analogs have pairing properties so they can produce ...... - One base analog widely used is 2-amino-purine (2-AP) which is ......

- base analogs - mutations by causing incorrect nucleotides to be inserted opposite them in replication → the original base analog exits in only a single strand but it can cause nucleotide-pair substitution that is replicated in all DNA copies descended from the original strand - an analog of adenine that can pair with thymine or cytosine when pronated

Spontaneous Mutations in Humans: trinucleotide-repeat diseases: - DNA sequence analysis reveals the gene mutations contributing to many human hereditary diseases: many are from .....

- base-substitution or single-base-pair indel mutations but some are more complex → human disorders are sometimes due to duplication of short repeated sequences

The Ames test: evaluating mutagens in our environment: - Many compounds are potential .... and we need a valid model system to evaluate these compounds if they are safe or not - Bruce Ames recognized a strong correlation between ability of compounds to cause cancer and their ability to cause mutations: he said that measurement of mutation rates in bacterial systems would be an effective model for ...... → treated special strains of bacteria with extracts of rat livers containing metabolic enzymes → bacterial strains had one of several mutant alleles of a gene responsible for histidine synthesis that were known to "revert" only by ...... → the treated bacteria were exposed to the test compound then grown on petri plates containing ...... → Absence of histidine ensured that ........ → # of colonies on each plate and total # of bacteria tested were determined and frequency of reversion was measured → compounds that yielded metabolites inducing elevated levels of reversion relative to untreated control liver extracts would then clearly be ......

- cancer-causing agents (carcinogens) - evaluating the mutagenicity of compounds as a first level of detection of potential carcinogens - certain kinds of additional mutational events - medium lacking histidine - only revertant individuals containing the appropriate base substitution or frameshift mutation would grow - mutagenic and possible carcinogens

Error-prone repair: translesion DNA synthesis: - These mechanisms evolved to prevent the occurrence of potentially more serious outcomes such as ..... - A stalled replication fork can initiate cell-death pathway but these replication blocks can be bypassed by ..... → in E.coli this process requires that activation of the SOS system: ......

- cell death or cancer - the insertion of nonspecific bases - this system is induced as an emergency response to prevent cell death in the presence of significant DNA damage, SOS induction is a mechanism of last resort, a form of damage tolerance that allows the cell to trade death for a certain level of mutagenesis

In these diseases the trinucleotide repeats fall within the open reading frames of the transcripts of the mutated gene leading to ........ → its easy to understand why these diseases result from expansions of codon-size units 3 base pairs long

- expansions or contractions of the number of repeats of a single amino acid in the polypeptide

Deamination can lead to other problems for bacteria and eukaryotes: by analyzing large # of mutations in the lacI gene, MIller identified places in the gene where one or more bases were prone to ........ → DNA sequence analysis of G-C → T-A transition hot spots in the lacI gene showed that ....... are present at each hotspot → the height of each bar on the graph represents ........ → the positions of 5-methylcytosine residues correlate with ......

- frequent mutation and these mutational hotspots corresponded to deamination at certain cytosine residues - 5-methylcytosine residues - the frequency of mutations at each site - most mutable sites

How do scientists know when all the components of a biological pathway have been identified? - All components of NHEJ pathway have been identified however geneticists analyzed a cell line (called 2BN) derived from a child with rare inherited disorder → they showed that cell line 2BN was defective for double-strand-break repair but they couldn't restore the repair system and produce the wild-type phenotype by ....... → when they introduced wild-type genes encoding known NHEJ proteins into the 2BN line ....... (what happens)?

- genetic complementation with any of the genes encoding NHEJ proteins - the cell line was still defective in the repair of double-strand breaks and this negative result shows that cell line 2BH carried mutation in unknown NHEJ protein

Both stalled replication forks and stalled transcription complexes activate this repair pathway which shows there are 2 types of nucleotide-excision repair that differ in damage recognition: Type 1: - global genomic nucleotide-excision repair (GG-NER): Type 2: - transcription-coupled nucleotide-excision repair (TC-NER):

- global genomic nucleotide-excision repair (GG-NER) - corrects lesions anywhere in the genome and is activated by stalled replication forks - transcription-coupled nucleotide-excision repair (TC-NER) - repairs transcribed regions of DNA

Higher eukaryotes put double stranded broken ends back together by nonhomologous end joining (NHEJ): - 1st step) recognize the damage, NHEJ pathway is initiated when 2 very abundant proteins, KU70 and KU80, bind to the broken ends forming a ...... that serves 2 functions: ....... → DNA ligase IV then joins the 2 ends

- heterodimer - 1st) it prevents further damage to the ends & 2nd) it recruits other proteins that trim the strand ends to generate the 5'-P and 3'-OH ends that are required for ligation

The target of the human mismatch system is short repeat sequences that can be expanded or deleted in replication by the slipped-mispairing mechanism → mutations in some parts of this pathway are responsible for many ...... → there are thousands of short repeats (microsatellites) located throughout the human genome and most are located in ..... - Defects in human mismatch-repair pathway would be predicted to have ....... → ex: hereditary nonpolyposis colorectal cancer (HNPCC) which is not cancer itself but increases cancer risk → have shown that HNPCC results from loss of mismatch-pair system due to inherited mutations in genes that encode the human counterparts (and homologs) of the bacterial MutS and MutL proteins → inheritance of HNPCC is autosomal dominant: cells with one functional copy of the mismatch-repair gene have ...... but tumor cell lines arise from ......

- human diseases especially cancers - noncoding regions but a few are located in genes that are critical for normal growth and development - very serious disease consequences - normal mismatch-repair activity - cells that have lost the one functional copy and are thus mismatch deficient and these display high mutation rates owing to inability to correct formation of indels in replication

The molecular consequences of point mutations in a coding region: - Synonymous mutations: - Missense mutations:

- mutation changes one codon for an amino acid into another codon for that same amino acid, synonymous mutations in exons are also called silent mutations (have no effect); they Never change the amino acid sequence of the polypeptide chain - codon for one amino acid is changed into a codon for another amino acid, they are also called nonsynonymous mutations

Luria-Delbruk fluctuation test - Analysis of mutations in bacteria that confer resistance to specific environmental agents ..... - Experiment: if E. coli bacteria are spread on a plate of nutrient medium in the presence of phage T1, the phages soon infect and kill the bacteria → rarely colonies were seen resistant to ...... HOWEVER whether these mutants were produced spontaneously or not isn't known - If mutations occurred spontaneously, then the mutations might be expected to ..... → the number of resistant colonies per culture should show high variation (fluctuation) → ......

- not normally tolerated by wild-type cells - phage attacks and were stable/appeared to be genuine mutants - occur at different times in different cultures; jackpot mutation (spontaneous)

Transitions: each of the bases in DNA can appear in one of many tautomeric forms that can ........, mismatches can also result when one of the bases becomes ionized: ........→ if proofreading doesn't happen all the mismatches lead to transition mutation in which ...... → other repair systems correct many of the mismatched bases that escape correction by the polymerase editing function

- pair to the wrong base - occurs more frequently than mismatched due to tautomerization and are corrected by the proofreading function of bacterial DNA pol III - a purine substitutes for a purine or a pyrimidine substitutes for a pyrimidine

"Fluctuation test": they inoculated 20 small cultures each with a few cells and incubated them until there were 10^8 cells per mL and at the same time a much larger culture also was inoculated and incubated until there were 10^8 cells per mL → the 20 individual cultures and 20 samples of the same size from the large culture were plated in the presence of ...... → the 20 individual cultures showed .......: 11 plates had 0 resistant colonies and other 9 have different numbers of resistant colonies → 20 samples of large culture showed ..... - If the phage were inducing mutations, there was no reason by fluctuation should be higher on individual cultures bc all were exposed to phage similarly → showed that ...... → led to the "paradigm" of mutation =

- phage - high variation in number of resistant colonies - much less variation in plates - the mutation was occurring randomly in time:the early mutations have higher numbers of resistant cells bc the mutant cells ahd time to produce many resistant descendants AND the later mutations produced fewer resistant cells - whether in viruses, bacteria, or eukaryotes mutations can occur in any cell at any time and their occurrence is random and spontaneous

Aflatoxin B1: - this addition product leads to breakage of bond between base and the sugar which ...... - aflatoxin B1 is a member of a class of chemical carcinogens known as .....

- powerful carcinogen that attach to guanine at the N-7 position - liberates the base and generates an apurinic site - bulky addition products when they bind covalently to DNA

Autosomal recessive disorder called xeroderma pigmentosum: ...... → mutated genes to develop this disease phenotype → people with this disease are examples of genetic variants: ........., how do these variants arise?

- prone to developing skin cancers and VERY sensitive to sunlight - individuals that show phenotypic differences in one or more particular character is

1st step in mismatch-repair is ...... → binding of this protein to distortions in the DNA double helix caused by mismatched bases initiates the mismatch-repair pathway by attracting ..... → key protein is MutH which ....

- recognition of the damage in newly replicated DNA by the MutS protein - 3 other proteins to the site of the lesion - cuts the strand containing the incorrect base by replacing the base on that strand

Several bypass polymerases that appear to be always present in eukaryotic cells are now known: they are always present so their access to DNA must be ..... → integral part of the replisome is the PCNA (proliferating cell nuclear antigen) protein that ..... - One protein present at stalled replication fork is Rad6, which is ...... but the binding of single ubiquitin monomer to PCNA changes its confirmation so it can now ..... → enzymatic removal of the ubiquitin tag on PCNA leads to dissociation of the bypass polymerase and restoration of normal replication → any base mismatch due to translesion synthesis still has .....

- regulated so that they are used only when needed - functions as a sliding clamp to orchestrate the myriad events at the replication fork - Rad6: an enzyme that adds ubiquitin to proteins (addition of chains of many ubiquitin monomers targets a protein for degradation) - bind the bypass polymerase and orchestrate translesion synthesis - change of detection and correction by the mismatch-repair pathway

Base-excision repair: exploit properties of antiparallel complementarity to restore damaged DNA segments to their initial, undamaged state → a base of longer segment of DNA chain is ....... → these pathways include the ..... - After DNA proofreading by DNA polymerase, base-excision repair is most important mechanism used to remove incorrect or damaged bases, the main target is ..... and this type of damage can result form variety of causes including ..... - Base-excision repair is done by DNA glycosylases that ...... → enzyme called PA endonuclease nicks the damage strand upstream of the AP site → 3rd enzyme called deoxyphosphodiesterase cleans the backbone by ....... → DNA ligase then ......

- removed and replaced with newly synthesized nucleotide segment complementary to the opposite template strand; removal and replacement of one or more bases - nonbulky damage to bases; methylation, deamination, oxidation, or spontaneous loss of DNA base - cleave base-sugar bonds and liberate altered bases and generate apurinic or apyrimidinic (AP) sites - removing stretch of neighboring sugar-phosphate residues so that a DNA polymerase can fill the gap with nucleotides complementary to the other strand - seals the new nucleotide into the backbone

2 autosomal recessive human diseases: xeroderma pigmentosum (XP) (........) and Cockayne syndrome (.........) are caused by defects in ......

- sensitive to UV light and get cancer early - dwarfism, deafness, and retardation, age prematurely - nucleotide-excision repair

How does mismatch repair distinguish the newly synthesized strand from the old one? - E.coli DNA is methylated but the methyl groups relevant to mismatch repair are added to adenine bases and uses the delay in methylation of the following sequence: 5' - G-A-T-C - 3' 3' - C-T-A-G - 5' - The methylating enzyme is adenine methylase which creates the 6-methyladenine on each strand → adenine methylase requires several minutes to recognize and modify the newly synthesized FATC stretches and in this interval the MutH protein nicks the methylation site on the strand containing the A that has not yet been methylated → this site can be ...... → after the site has been nicked the ......→ Protective single-strand-binding protein ....... - Many of the proteins in E.coli mismatch repair are conserved in .....

- several hundred base pairs away from the mismatched base - UrvD protein binds at the nick and uses its helicase activity to unwind the DNA - coats the unwound parental strand while the part of the new strand between the mismatch and the nick is excised - human mismatch repair

Ionizing radiation results in formation of ionized and excited molecules that can damage DNA → due to aqueous nature of biological systems ...... → different types of reactive oxygen species are produced but the most damaging to DNA bases are ......

- the molecules generated by ionizing radiation on water produce the most damage - OH, O2-, and H2O2 which lead to formation of different adducts and degradation products

The closer the nonsense mutation is to the 3' end of the open reading frame (ORF) the more likely it is that .... - Many nonsense mutations produce .... - Single-base-pair changes that inactivate proteins are often due to ..... → significantly change the mRNA transcript by leading to .....

- the resulting protein might have some biological activity - completely inactive protein products - splice site mutations; large insertions or deletions that may or may not be in frame

Regulation of PCNA function by the addition and removal of ubiquitin monomers shows the importance of post-translational modifications in eukaryotes → if base damage in the template strand is not corrected quickly then ..... → eukaryotic cell cannot wait for the de novo synthesis of bypass polymerases following transcription and translation as occurs in the E.coli SOS system but instead eukaryotic bypass polymerases are ......

- the stalled replication fork will signal activation of cell-death pathway - transcribed and always present, their access to the replication fork is controlled by rapid and reversible post-translational modifications

What can geneticists do in the lab to find a protein, such as the unknown NHEJ protein, that has not yet been identified? - Protein is NHEJ component XRCC4 and the ...... identified a 33-kD interacting protein that was encoded by an uncharacterized human open reading frame → the interaction of 2 proteins in the yeast two-hybrid test does not mean these proteins interact in human cells, to establish connection between 33-kD protein and NHEJ pathways RNAi technique is used: ..... (showed what??) - 2BN cells defective in double-strand repair lack the 33-kD protein and expression of this protein does what???

- two-hybrid test - using RNAi, it showed that normal cells expressing antisense RNA from the ORF that encodes the 33-kD proteins which would prevent translation of this gene into protein were not defective in the execution of NHEJ pathway - corrects the cellular defects

Patients with Cockayne syndrome have mutation in ....

1 of 2 proteins called CSA and CSB which recognize stalled transcription complexes

Patients with XP fall into 8 complementation groups carrying mutations in .....

1 of 8 genes encoding proteins XPA through XPG

Mechanisms of spontaneous mutations: - Spontaneous mutations arise from variety of sources: 1) DNA-replication process: 2) Can also arise bc DNA is a very labile molecule and the ..... itself can damage it 3) Mutations can also be caused by the insertion of .....

1) DNA replicated is an accurate process but mistakes are made in the copying of million/billion of base pairs in a genome 2) cellular environment 3) transposable element from elsewhere in the genome

Mechanisms of mutagenesis: mutagens induce mutations by at least 3 different mechanisms: - Mutagenizing genes and observing phenotypic consequences

1) Replace a base in the DNA 2) alter a base so it specifically mispairs with another base 3) damage a base so it can't pair with any base under normal conditions anymore

These translesion, or bypass, polymerases: differ from the main replicative polymerases in several ways: - 1st) they can tolerate unusually ...... whereas the replicative polymerase stalls if a base doesn't fit into an active site, the bypass polymerases have - 2nd) in some situations the bypass polymerases have a much higher error rate because - 3rd) they can only add a few nucleotides before falling off which is good because the main function of an error-prone polymerase is to .....

1) large adducts on the bases; much larger pockets that can accommodate damaged bases 2) they lack the 3'-5' proofreading activity of the main replicative polymerases 3) unblock the replication fork instead of synthesizing long stretches of DNA that could contain many mismatches

Repair of double-strand breaks: many correction systems exploit DNA complementarity to make error-free repairs: 2 stages: - Ex: exposure to X-rays often cause both strands of double helix to break at sites that are close together = double-strand break → if left unrepaired ..... - Generation of double-strand breaks is an integral feature of some .... - Ex: meiotic recombination when the cell uses many of the same proteins and pathways to repair double-strand breaks and to carry out meiotic recombination → ......

1) removal of the damaged bases, along with nearby DNA, from one strand of the double helix 2) use of the other strand as a template for the DNA synthesis needed to fill the single-strand gap but this would happen if both strands of the double helix were damaged in a way that complementarity could not be exploited - the double-strand breaks can cause a variety of chromosomal aberrations resulting in cell death or a precancerous state - normal cellular processes that require DNA rearrangements - focus on the molecular mechanisms that repair double-strand breaks before focusing on meiotic recombination

nucleotide-excision repair requires dozens of proteins, and has 4 phases:

1. Recognition of damaged base(s) 2. Assembly of a multiprotein complex at the site 3. Cutting of the damaged strand several nucleotides upstream and downstream of the damage site and removal of the nucleotides (ab 30) between the cuts 4. Use of the undamaged strand as template for DNA polymerase followed by strand ligation

Mismatch repair systems have to do at least 3 things:

1. Recognize mismatched base pairs 2. Determine which base in the mismatch is the incorrect one 3. Excise the incorrect base and carry out repair synthesis

1st steps in SDSA are ...... by specialized proteins and enzymes, the trimming of the 5' ends by an ...... , and the coating of these regions with proteins that include the ...... → recall in SOS response RecA monomers associate with regions of single-stranded DNA to form nucleoprotein filaments and Rad51 forms long filaments as it associated with the exposed single-stranded region → Rad51-DNA filament takes part in remarkable search of the undamaged sister chromatid for complementary sequence that will be used as a template for DNA synthesis: this process is called strand invasion: ....... → new DNA synthesis continues from both 3' ends until both strands unwind from their templates and anneal → ligation seals the nicks leaving a repaired path of DNA that has been replicated by a conservative process → both strands are newly synthesized strands in marked contrast to the semiconservative replication of most DNA

1st) binding of broken ends 2nd) endonuclease to expose single-stranded regions 3rd) RecA homolog, Rad51 - strand invasion: the 3' end of the invading strand displaces one of the undamaged sister chromatids which forms a D-loop, and primes DNA synthesis from its free 3' end

mutagens

A chemical or physical agent that interacts with DNA and causes a mutation.

translesion DNA synthesis

A damage-tolerance mechanism in eukaryotes that uses bypass polymerases to replicate DNA past a site of damage

Ames test:

A procedure using bacteria to identify potential carcinogens provided a way to screen thousands of compound and evaluate one aspect of their risk to health and the environment

Nonhomologous end joining: - cells of higher eukaryotes are usually not replicating their DNA bc .....

A quick-and-dirty mechanism for repairing double-strand breaks in DNA that involves quickly bringing together, trimming, and rejoining the two broken ends; results in a loss of information at the site of repair (prone to error) - they are either in resting phase of cell cycle or have ceased dividing entirely

Nucleotide-excision repair: - This mechanism cannot correct ...... that discord the DNA helix, adducts such as the cyclobutane pyrimidine dimers caused by UV light, and cannot correct damage to ...... - A DNA polymerase cannot continue DNA synthesis past such lesions and the result is ..... - Abnormal or damaged base can stall the .... - To fix all of these problems: prokaryotes and eukaryotes use pathway called nucleotide-excision repair (NER):

A repair system that removes and then correctly replaces a damaged segment of DNA using the undamaged strand as a guide - bulky adducts; more than one base - replication block → blocked replication fork can cause cell death - transcription complex - able to relieve replication and transcription blocks and repair the damage

Homologous recombination: homologous recombination repair: - if double strand break occurs after replication of chromosomal region in dividing cell, the damage can be corrected by error-free mechanism called ..... - synthesis-dependent strand annealing (SDSA):

Exchange of genetic information between homologous DNA molecules - uses sister chromatid to repair the break - synthesis-dependent strand annealing (SDSA) - uses sister chromatids available in mitosis as the templates to ensure correct repair

The Molecular Basis of Induced Mutations:

Induced mutations are not spontaneously but rather caused by a mutation present in the environment whether intentionally in the lab or accidentally encountered in everyday life

Why are 5-methylcytosine hot spots for mutations? - Thymine is not recognized by the enzyme uracil-DNA glycosylase and is not repaired so C→T transitions generated by deamination are seen more frequently at ...... → consequence of frequent mutation of 5-methylcytosine to thymine is that .....

The deamination of 5-methylcytosine generates thymine (5-methyluracil) - 5-methylcytosine sites bc they escape this repair system - methylated regions of the genome are converted to AT-rich regions and in contrast, coding and regulatory regions (which are less methylated) remain GC rich

What happens when double-strand breaks occur in cells where undamaged strands or sister chromatids are not present?

These ends can initiate potentially harmful chromosomal rearrangements that could lead to a cancerous state

heterodimer

a chain of two molecules that are different from each other

Errors in DNA replication:

a mistake by DNA polymerase may cause a point mutation Can result when an illegitimate nucleotide pair (ex: A-C) forms in DNA synthesis, leading to a base substitution that may be either a transition or transversion → other errors may add or subtract base pairs and create a frameshift mutation

Cockayne syndrome patients cant recognize stalled transcription complexes → cell more likely to .....

active apoptosis suicide pathway

- Oxidatively damaged bases represent a 3rd type of spontaneous lesion that makes mutations:

active oxygen species such as superoxide radical, hydrogen peroxide, and hydroxyl radicals, are produced as by-products of normal aerobic metabolism and can cause oxidative damage to DNA and to precursors of DNA (such as GTP) resulting in mutations → mutations from oxidative damage have been seen in human diseases

The Phenotypic Consequences of DNA Mutations: - Point mutation: ....... → what are the effects of this at phenotypic level?

alteration of a single base pair of tDNA or of a small number of adjacent base pairs

Induced mutations:

arise through action of certain agents called mutagens that increase the rate at which mutations occur

Ionizing radiation can also damage DNA directly rather than through reactive oxygen species → this way causes .....

breakage of N-glycosidic bond leading to formation of apurinic or apyrimidinic sites and can cause strand breaks → strand breaks are responsible for most lethal effects of ionizing radiation

Many of these diseases include neurodegeneration:

cell death within the nervous system

Recombination is the outcome of ....

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

Specific mispairing: - some mutagens are not incorporated into the DNA but instead alter a base in a way that it'll form a specific mispair → certain alkylating agents such as ........ operate in this pathway - These agents add alkyl groups (an ethyl group in EMS and methyl group in NG) to many positions on all 4 bases and the formation of a mutation is best correlated with an addition to the oxygen at position 6 of guanine to create an O-6-alkylguanine → leads to ....... - Alkylating agents can also modify the bases in .....

chemicals that alter a base, causing it to have unusual pairing - ethyl methanesulfonate (EMS) and nitrosoguanidine (NG) - direct mispairing with thymine and would result in G-C → A-T transitions at the next round of replication - dNTPs (N is any base) which are precursors in DNA synthesis

Nonsense mutations:

codon for one amino acid is changed into a translation-termination (stop) codon → lead to the premature termination of translation so it has a large effect on protein function

2 types of missense mutations

conservative and nonconservative

2 of the most frequency spontaneous lesions result from .....

depurination or deamination

GG-NER is initiated when protein complex of XPC and RAD23B recognizes a ..... - TC-NER is initiated when an RNA polymerase complex is stalled by DNA lesion in the transcribed strand and CSA and CSB bind at this site to form a ...... → after lesion recognition, the GG-NER and TC-NER pathways use the same proteins to ...... bc the role of XPC-RAD23B and SA/CSB is to ......: 2 of TFIIH complex subunits XPB and XPD are helicases (3'-5' and 5'-3') that ...... → subsequent steps common to GG-NER and TC-NER mediate the cleavage and excision of the damaged base and as many as ...... → in addition of XPC-RAD23D, XPB, and XPD, XP patients have mutations in other proteins that participate in the common steps of NER: - XPA promotes release of the CAK subunit and binding of RPA while endonuclease XPF and XPG cut 5' and 3' of the DNA damage → after removing damaged base and surrounding DNA, the gap is filled by ...... → last step of NER involves ...... by 1 of 2 ligation complexes (XRCC1/LIG3 pr FEN1/LIG1)

distorted double helix caused by damaged base and binds to the opposite strand - recognition complex; remove and repair the damaged DNA; attract the multiprotein TFIIH complex - unwind and open the DNA helix around the lesion - 30 adjacent nucleotides followed by DNA synthesis to fill the gap - DNA polymerase assisted by RFC and PCNA proteins - initiation of new strand to surrounding DNA

DNA molecules are not absolutely stable:

each base pair in DNA double helix has certain probability of mutating which could be swapping one base pair for another or the disappearance of an entire chromosomes

Trinucleotide-repeat diseases: - Ex: human disease called fragile X syndrome: most common form of inherited ...... and is manifested cytologically by a fragile site in the ..... → fragile X syndrome results from changes in the number of a ..... - Humans normally show variation in the number of CGG repeats in the FMR-1 gene ranging from 6-54 and sometimes unaffected parents and grandparents give rise to several offspring with fragile X syndrome → the offspring have ..... → the unaffected parents and grandparents have increased copy numbers of the repeat but ranging from only 50-200 → the ancestors have been said to carry permutations: ..... - Generation of these repeats is from .... - In humans cells after a threshold level of ab 50 repeats, .....

expansion of a 3-base-pair repeat - mental impairment; X chromosome that results in breaks in vitro - CGGn repeat in a region of the FMR-1 gene that is transcribed but not translated - enormous repeat numbers ranging from 200-1300 - the repeats in these permutation alleles are not sufficient to cause the disease phenotype but they are more unstable than normal alleles so they lead to even greater expansion in progeny - slippage that occurs in course of DNA synthesis - the replication machinery cannot faithfully replicate the correct sequence and large variations in repeat numbers result

oxidatively damaged bases - Ex: the 8-oxo-7-hydroxydeoxyguanosine product frequently mispairs with A resulting in .....

free radicals in the cell can chemically alter nitrogenous bases and cause subsequent mispairing - high level of G-T transversions → The thymidine glycol product blocks DNA replication if unrepaired

point mutation

gene mutation in which a single base pair in DNA has been changed

We are familiar with DNA damage induced by UV light and an unusual class of E.coli mutants that survived UV exposure without sustaining additional mutations was isolated and some E.coli genes function to ...... → the UV-light induced mutation will not occur if the DinB, UmuC, or UmuD' genes are mutated

generate mutations when exposed to UV light

Some replication errors lead to indel mutations:

insertion-deletion of one or more base pairs that produce frameshift mutations when they add or subtract a number of bases not divisible by 3 (size of codon) in the protein-coding regions

Depurination: - Mammalian cell spontaneously loses ab 10,000 purines form its DNA in a 20-hr cell-cycle period at 37 degrees Celsius and if these lesions continued they would result in ....... → efficient repair systems remove apurinic sites and a base can be inserted across from an apurinic site which results in ....

loss of a purine base, consists of the interruption of the glycosidic bond between the base and deoxyribose and the subsequent loss of a guanine or an adenine residue from the DNA → the DNA backbone remains intact - a significant genetic damage bc the resulting apurinic sites cannot specific a base complementary to the original purine; mutation

base insertion

mutation which results in the addition of nucleotide pairs in a gene; have a major effect on the resulting protein

base deletion

mutation which results in the loss of nucleotide pairs in a gene; have a major effect on the resulting protein

Gene mutations: - Mutations lead to .... - Most serious DNA damage is a .... - ..... and ..... are major sources of variation - Mechanisms of ..... and ..... have features in common

mutational events happening within individual genes - evolution - double-strand break - Mutation and recombination - DNA repair and recombination

XP patients can recognize stalled transcription complexes (they have normal CSA and CSB proteins and prevent cell death when transaction is restarted but they can't repair the original damage because of mutation in one of their XP proteins → ......

mutations will accumulate in the cells of patients with XP and the presence of mutations whether caused by mutagens or failure of repair pathways increases risk for developing cancers

Spontaneous lesions:

naturally occurring damage to DNA that generate mutations

Spontaneous mutations:

naturally occurring mutations and arise in all cells

Repairing double stranded breaks: (2 ways)

nonhomologous end joining and homologous recombination

Nonconservative substitution:

one amino acid may be replaced by a chemically different amino acid → this alteration is more likely to produce a severe change in protein structure and function

UV light usually causes damage to nucleotide bases: it generates a number of distinct type of alterations in DNA called ...... which lead to .....

photoproducts mutations of 2 different lesions that unite adjacent pyrimidine residues in the same strand → lesions are the cyclobutane pyrimidine photodimer and the 6-4 photoproduct

Transversions:

pyrimidines substitute for a purine (vice versa), creation of transverion by replication error requires the mispairing of a purine with a purine or a pyrimidine with a pyrimidine

Transition:

replacement of a base by the other base of the same chemical category → either a purine is replaced by another purine (G & A) or a pyrimidine is replaced by another pyrimidine (C & T)

Transversion:

replacement of a base of one chemical category buy a base of the other → either a purine into a pyrimidine or a pyrimidine into a purine

Base damage: large number of mutagens damage one or more bases and results in .....

replication block so DNA synthesis will not proceed past a base that cannot specify its complementary partners by H-bonding → replication blocks can cause more mutation

Frameshift mutations:

replication errors lead to these which create greatly changes proteins

This suggest that resistant cells are ....

selected by the environmental agent (phage) rather than produced by it

Double-strand breaks can arise .....

spontaneously or can be induced by ionizing radiation

Human mismatch-repair system was reconstituted in the test tube in the Modrich lab → the ability to study details of the reaction will ....

spur future studies of the human pathway

Base insertions or deletions: insertions or deletions of nucleotide pairs = indel mutations (for insertion-deletion), .....

the addition or deletion of a single base pair is the simplest mutation here

Deamination: - Deamination of cytosine yields uracil → unpaired uracil residues will pair with adenine in replication resulting in ....

the removal of an amino group from an organism, particularly from an amino acid - the conversion of a G-C pair into an A-T pair

Huntington's disease with expansion of trinucleotide repeats in a gene or its regulatory regions:

the wild-type HD gene includes a repeated sequence often within the protein-coding region and a mutation correlates with expansion of this repeat region and the severity of the disease correlates with the number of repeat copies

Huntingtons and kennedy disease (progressive muscle weakness and atrophy) result from the amplification of 3-base-pair repeat CAG → .....

unaffected people have ab 19-21 CAG repeats and affected have ab 46

Conservative substitution:

when the mutation replaces one amino acid with a chemically similar amino acid → the alteration is less likely to affect the proteins structure and function severely