Molecular Biology: DNA, RNA, and Protein Synthesis, Exams of Medical Biochemistry

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Medical Biochemistry w/ Borgon Exam 4

Review

A nucleotide has a ___________________ base, a _________ (β-furanose), and a phosphate. - ANSWERS: Nitrogenous; ribose A _________________ has a base and sugar. - ANSWERS: Nucleoside Nucleotides have a variety of roles in cellular metabolism, and function as ___________ (ATP), ______________ (NAD+, FAD), _____________ (cAMP), and as ________________ in intercellular communication. - ANSWERS: Energy; cofactors; signaling; messengers Nucleotides can polymerize into ___________ ___________. - ANSWERS: Nucleic acids Note: The basis of the structure of every protein, and ultimately of every biomolecule and cellular component, is a product of information programmed into the sequence of nucleic acids. The ability to store and transmit genetic information from one generation to the next is a fundamental condition for life. Nucleic acids __________ genetic information (DNA), ___________ genetic information (mRNA), ________________ genetic information (ribozymes), and ____________ ______________ (rRNA and tRNA). - ANSWERS: Store; transmit; process; synthesize proteins What are the purines? - ANSWERS: Adenine (A) and Guanine (G) What are the pyrimidines? - ANSWERS: Cytosine (C), Thymine (T), and Uracil (U) True or false: Purines and pyrimidines are neutral at pH 7. - ANSWERS: True What are prototropic tautomers? - ANSWERS: Structural isomers that differ in the location of protons

_______/_________ conformations are the most common, and uncommon tautomers can lead to ___________________. - ANSWERS: Amino/keto; mispairing Which nitrogenous bases have the amino conformation? - ANSWERS: Cytosine and adenine Which nitrogenous bases have the keto conformation? - ANSWERS: Guanine and thymine Nucleotides can be joined via __________________ ______________, which gives the backbone a _______________ charge. This results in linear polymers with no branching or ________________. - ANSWERS: Phosphodiester linkages; negative; crosslinks Note: The directionality is 5' -> 3' How is the DNA backbone very stable? - ANSWERS: The 2'-H, which helps it survive thousands of years Why is RNA unstable? - ANSWERS: The 2'-OH Note: RNA is unstable and lasts a few years in water, but only a few hours in cells. It is unstable under alkaline conditions or when hydrolyzed by enzymes (RNase). Two bases can ____________ bond to form a base pair. - ANSWERS: Hydrogen Watson-Crick base pairs predominate in double-stranded DNA, where purines pair with ___________________ based on the H-bonds. - ANSWERS: Pyrimidines Determine if the following characteristics correctly describe the DNA structure: A. Double helix B. Sugar/phosphate (+ charge)

C. Deoxyribose D. Base pairing E. Parallel F. Has a major and minor groove G. 2 nm wide H. 3.4 nm/ 10.5 bp/turn (variable) - ANSWERS: A. True B. False C. True D. True E. False F. True G. True H. True Match the following: A. A-DNA B. B-DNA C. Z-DNA

1. Most DNA
2. Dehydrated
3. Left-handed (syn purines) - ANSWERS: A- 2 B- 1 C- 3 DNA is stabilized by H-bonds and ______ __________ (pi stacking), which are van der Waals interactions that result from the _________________ effect (excluding water), as well as pi bonds overlapping (_____________ _____________). - ANSWERS: Base stacking; hydrophobic; charge penetration

In DNA replication, the strands _______________ and each serves as a ______________ for synthesis of a new strand. This is catalyzed by _____ ________________. The new DNA molecule has one parent strand and one _______________ strand. - ANSWERS: Separate; template; DNA polymerases; daughter True or false: Mutations can also arise during replication. - ANSWERS: True How can the bases be damaged? - ANSWERS: By depurination (hydrolysis of glycosidic bond), deamination, oxidation, methylation, UV light, and X-rays RNA is similar to DNA, but different in several ways. Determine if the following is true or false: A. H replaces an OH at 2' carbon of sugar B. Uracil: absence of CH3 compared to thymine C. Single stranded, sometimes double stranded (A form, not B form) but irregular D. Adapter between DNA and amino acids E. Genetic material F. Cannot have secondary and tertiary structure G. Divalent metals like Mg2+ often bind to shield charges, allow tight packing H. Unique bases and base pairs - ANSWERS: A. False B. True C. True D. True E. False F. False G. True H. True RNA is synthesized using a _______ template (with some exceptions), and it contains a 2'-OH and uracil. - ANSWERS: DNA

RNA can fold into __________ (ribozymes). - ANSWERS: Enzymes RNA, like proteins, will fold to _______________ weak interactions and find the _________________ free energy. - ANSWERS: Maximize; minimum Nucleotide pools are kept low, so cells must continually synthesize them in the __________ (mostly in the _____________, but some pyrimidine steps are in the ____________________). - ANSWERS: Liver; cytoplasm; mitochondria What are nucleotides synthesized from? - ANSWERS: Amino acids, ribose 5-phosphate, CO2, and NH ___________________ provides most amino groups, _____________ is the precursor for purines, and __________________ for pyrimidines. - ANSWERS: Glutamine; glycine; aspartate Nucleotides can be ____________ from nucleobases. - ANSWERS: Salvaged Note: Antiparasitic drugs often target the salvage pathway since parasites such as malaria cannot synthesize them. Where is ribose 5-phosphate made? - ANSWERS: In the pentose phosphate pathway Where are purines synthesized? - ANSWERS: On the ribose Note: Inosine-P -> AMP/GMP Unlike purine synthesis, pyrimidine synthesis proceeds by first making the _______________ ________ and then attaching it to __________ _______________ (phosphoribosyl pyrophosphate, PRPP). - ANSWERS: Pyrimidine ring; ribose 5-phosphate How is the first committed step of pyrimidine synthesis catalyzed? - ANSWERS: By aspartate transcarbamoylase (ATCase)

Note: This is inhibited by CTP, and activated by ATP Ribonucleotide reductase catalyzes the reduction of ribonucleotides to _____________________________. - ANSWERS: Deoxyribonucleotides Note: 2'C-OH bond is directly reduced to 2'-H bond, without activating the carbon (uses free radicals) Another note: This is inhibited by dATP. ______________ can be made from uracil or cytosine by thymidylate synthase (anticancer target), which adds a methyl group from _________________________. - ANSWERS: Thymine; tetrahydrofolate ________ ______ supplements are taken during pregnancy to ensure biosynthesis can occur. Deficiency is widespread, especially in nutritionally poor populations. - ANSWERS: Folic acid Reduced thymine synthesis causes ___________ to be incorporated into DNA, and the repair mechanism can cause DNA _________ _________, and is associated with cancer, heart disease, and neurological impairment. - ANSWERS: Uracil; strand breaks Match the following antibiotics and anticancers with the correct function: A. Allopurinol B. Trimethoprim/sulfamethoxazole C. Methotrexate and aminopterin D. Azaserine and acivicin E. Fluorouracil

1. Glutamine analogs that inhibit glutamine amidotransferases, antibiotic/anticancer
2. Inhibit bacterial dihydrofolate reductase and dihydropteroate synthase

3. Is converted by the salvage pathway into FdUMP, which inhibits thymidylate synthase, antibiotic/anticancer
4. Used against trypanosomes which lack enzymes for nucleotide synthesis
5. Inhibit dihydrofolate reductase, and force cancer cells to use the salvage pathway - ANSWERS: A- 4 B- 2 C- 5 D- 1 E- 3 In nucleotide catabolism, Purines are __________________, deaminated to ___________, and oxidized into ________ ________. - ANSWERS: Dephosphorylated; xanthine; uric acid High concentrations of uric acid can lead to ______, which are uric acid crystals in joints, especially the toes, and higher rates of diabetes and kidney stones. - ANSWERS: Gout How is gout treated? - ANSWERS: With diet changes and allopurinol, which inhibits xanthine oxidase Free bases can be salvaged, and lack of ____________________-_____________ __________________________________ leads to Lesch-Nyhan Syndrome, which results in neurological impairment, and finger/toe biting. - ANSWERS: Hypoxanthine-guanine phosphoribosyl transferase (HPRT) Humans have the urate oxidase (___________) gene, but lost its function in several primates 9-15 million years ago. - ANSWERS: Uricase Uric acid is an ________________, and it may counter the loss of the vitamin C gene. - ANSWERS: Antioxidant True or false: Laboratory expression in human liver cells showed a lower fructose to triglyceride conversion, so loss of the enzyme could have helped during times of starvation. - ANSWERS: True

Note: It may have additional impact on dopamine, immune function, and salt sensitivity, and blood glucose. With modern food excess, ______ _______ may lead to obesity, gout, insulin resistance, hypertension, kidney stones, and cardiovascular disease. - ANSWERS: Uric acid Pyrimidine catabolism leads to NH4+, CO2, and then _______. - ANSWERS: Urea Breakdown products in pyrimidine catabolism can also be used in the ____________ __________ __________. - ANSWERS: Citric acid cycle Where does DNA absorb UV light? - ANSWERS: 250-270 nm, with a peak at 260 nm DNA can also denature, which increases base absorbance due to loss of _______ ___________________. This can be analyzed in a spectrophotometer to determine a sequence's _______________ ________________, the point at which 50% is melted. - ANSWERS: What increases the melting temperature? - ANSWERS: Higher GC content and salt DNA can be separated on an __________________ gel via ______________________. - ANSWERS: Agarose; electrophoresis True or false: Negatively-charged DNA migrates to the - electrode in the presence of an electric field. - ANSWERS: False; Negatively-charged DNA migrates to the + electrode in the presence of an electric field. True or false: In DNA electrophoresis, larger molecules migrate more quickly. - ANSWERS: False; Smaller molecules migrate more quickly.

Note: Mobility depends on the size and conformation of DNA. What are agarose gels used for? - ANSWERS: DNA analysis, purification, and DNA interaction studies True or false: DNA can be sequenced with several different techniques. - ANSWERS: True Sanger sequencing uses ________ to terminate the chain. - ANSWERS: ddNTPs Genomes can be sequenced in hours with modern techniques called _____________________________________. - ANSWERS: Next-generation sequencing Note: The DNA is cut into small fragments and sequenced in parallel. There is no termination, thus it is not limited by the detection resolution of Sanger sequencing. _____________ can be sequenced and analyzed to study mutations, diseases, SNPs, ancestry, phylogenetic analysis between species, etc. - ANSWERS: Genomes DNA can be cleaved on its phosphate backbone with _________________ _______________. - ANSWERS: Restriction enzymes Note: One common example is EcoRI. Digest DNA fragments can be re-joined using ______ _____________. - ANSWERS: DNA ligase What are vectors? - ANSWERS: Small extrachromosomal pieces of circular DNA called plasmids that are found in bacteria, that can be cut and then ligated with another small piece of DNA (< 3000 bp) Note: DNA can be digested with a restriction enzyme and rejoined with DNA ligase.

__________________ _______ _______________ involves combining DNA sequences that do not occur together in nature. - ANSWERS: Recombinant DNA technology Note: This can be used to make plants resistant to glyphosate and insects, or to grow larger. Bacteria can express enzymes or compounds, animals can produce medicines and vaccines, and it is the basis of gene therapy. DNA sequences from two different sources (a genome and a vector) can be spliced together and inserted into a host cell, in a process called _______________ ______________. - ANSWERS: Molecular cloning Fill in the blanks for the steps of molecular cloning:

• Cut the source DNA with a __________________ _____________
• Insert the gene into a DNA ___________
• Insert recombinant vector into a host cell (__________________)
• Host produces multiple choices of recombinant DNA (_______________) - ANSWERS: Restriction enzyme; Vector; Transformation; Cloning True or false: The cloning process disrupts a cloning site which contains LacZ (β-galactosidase), which when expressed can cleave a galactose derivative to produce a white product. - ANSWERS: False; It produces a blue product. Note: The success of the cloning can be analyzed via blue/white screening. How can DNA be amplified? - ANSWERS: Polymerase chain reaction (PCR) Note: The region of interest can be copied many times.

The process of DNA amplification involves mixing target DNA with ____________ that are complementary to it, adding nucleotides, and a ______________ DNA polymerase. The sample is placed in a _______________ and copied _____ times. - ANSWERS: Primers; thermostable; thermocycler; 30 PCR can be analytically to detect differences in gene expression via __________________ ______. - ANSWERS: Quantitative PCR _____ __________________ show differences in gene expression. Microarray chips contain fragments from genes in the group to be analyzed, and can contain entire bacterial and yeast genomes. - ANSWERS: DNA microarrays What does analysis of DNA microarrays show? - ANSWERS: Which genes are present, tissue-specific expression, and cancer/disease analysis What are short tandem repeats (STRs)? - ANSWERS: Short sequences that repeat next to each other Note: These can be used in paternity testing or forensic crime scene analysis. Another note: Differences in the number of repeats cause variations in the length of fragments that form when a sample is subjected to PCR using a primer specific for that region. Eukaryotic genes can be processed from mRNA into ______ for expression in other systems (bacteria, yeast). - ANSWERS: cDNA Note: This can be placed into an expression vector which has promoter sequences, ribosome binding sites, and transcription termination sequences. The vector/cDNA can be transformed into bacteria, and proteins can be expressed (_____________). - ANSWERS: Induced Proteins can be purified on a column via ____________________, using affinity (tag or antibodies), charge, size, etc. - ANSWERS: Chromatography

Proteins can be analyzed using antibodies on a western blot for ___________________ or to determine their ________________________ state. - ANSWERS: Identification; phosphorylation Note: They can be run through many tests such as protein-protein interaction arrays, mutation analysis, binding affinity assays, drug interaction assays, etc. The __________________________________ screens thousands of proteins for interactions using yeast trasncription. - ANSWERS: Yeast two-hybrid assay True or false: Fluorescence can be used to determine the location of a protein within a cell. - ANSWERS: True Match the following: A. Green fluorescent protein (GFP) B. Immunofluorescence C. Pulse-chase

1. Tracks a metabolite through the cell
2. Can be tagged onto a protein and visualized under a fluorescent microscope
3. Detects proteins with antibodies - ANSWERS: A- 2 B- 3 C- 1 The discovery of DNA's __________ _________ ____________ of DNA in 1953 laid a foundation to thinking of biomolecules as carriers of information. - ANSWERS: Double helical structure What is the central dogma? - ANSWERS: DNA -> RNA -> Protein

Who proposed the central dogma? - ANSWERS: Francis Crick Historically, a gene was something responsible for a characteristic or ____________________. - ANSWERS: Phenotype Beadle and Tatum, through mutation studies on mold, defined a gene as something that coded for an ______________. - ANSWERS: Enzyme One gene -> one enzyme How are genes defined today? - ANSWERS: As segments of DNA that code for RNA or protein, and encodes multiple products In ___________________, one strand of double-stranded DNA acts as the molecular template for RNA synthesis. - ANSWERS: Transcription Note: DNA -> messenger RNA In ________________, the triplet codons of nucleotides in mRNA bind to complementary triplets in tRNA. - ANSWERS: Translation Note: The tRNA molecules contain amino acids associated with the particular triplet, and amino acids are assembled into peptides What does a protein's sequence determine? - ANSWERS: Biological function True or false: The linear dimensions of DNA are shorter than the virions or cells that contain them. - ANSWERS: False; The linear dimensions of DNA are longer than the virions or cells that contain them (bacteriophage T4 is 0.1x0.2 micrometer, DNA is 60 micrometers long); it requires ATP to package it.

Note: E. coli DNA (4.6 mbp) is 850x longer than the cell Another cell: DNA is organized into compact forms. True or false: Bacteria typically have multiple circular dsDNA chromosome. - ANSWERS: False; Bacteria typically have one circular dsDNA chromosome. DNA is found in ____________, where scaffold-like structure organizes the circular DNA into 500 looped domains. DNA can occupy much of cell volume and also attach to the plasma membrane. - ANSWERS: Nucleoid Note: DNA binds to DNA-binding proteins transiently. Human somatic cells are ___________ and have ___ chromosomes. - ANSWERS: Diploid; 46 The nuclear genome contains ___ pairs of chromosomes (_________) plus X and Y, variable size/length, linear, and packed around _______________. - ANSWERS: 22; diploid; proteins Note: 3 billion base pairs x 2 copies/cell, 37 trillion cells in the body; this amounts to 2 m of DNA per cell; there is only 0.5 grams of DNA in human body, but it would stretch 10 billion miles Mitochondria and ________________ were once free-living bacteria (________________ Theory), with their own circular dsDNA. - ANSWERS: Chloroplasts; Endosymbiotic Determine if the following describes human mitochondrial DNA (mtDNA), plant mtDNA, or chloroplast DNA: A. 500-10,000 mitochondria per cell B. Encodes its own tRNAs and rRNAs, and some proteins C. 120-160 kbp

D. 200 kbp- 2.5 mbp E. 16,569 bp, 2-10 copies per mitochondrion - ANSWERS: A. Human B. Human C. Chloroplast D. Plant E. Human Viruses contain ssRNA (+ or -), dsRNA, or dsDNA surrounded by _____________ coat, and use ___________ genes when they infect. - ANSWERS: Protein; host's Match the following: A. E. coli B. Humans C. Axolotl D. Lungfish

1. 32 billion bp, regrows bones, muscles, and nerves
2. 4,639,675 bp, 4400 genes for protein, 157 for catalytic or structural RNAs
3. 43 billion bp, hibernates up to four years and lives off tail muscle protein
4. 3 billion bp, 25,000 genes - ANSWERS: A- 2 B- 4 C- 1 D- 3 Note: Some plants have over 100 billion bp genomes. True or false: As genome size increases, complexity often increases. - ANSWERS: True

Does the total length of DNA or the number of chromosomes correlate with the complexity of an organism? - ANSWERS: No Note: Dogs have 78 chromosomes. Amphibians have much more DNA than humans. And plants have more genes than humans. Why is the correlation between genome size and complexity poor? - ANSWERS: Most of eukaryotic DNA is noncoding True or false: Recent experimental work suggests that a living organism could get by with less than 400 genes. - ANSWERS: True Note: Nasuia deltocephalinicola (endosymbiotic) is 112,091 base pairs with 137 genes. And Mycoplasma has 531,000 base pairs with 473 genes. What are exons? - ANSWERS: The small fraction (1.5%) of the total genome that encodes for proteins What are introns? - ANSWERS: Regions of genes that are transcribed but nor translated, and removed via splicing (all eukaryotes, only in some bacterial tRNA sequences) The biological significance of noncoding intergenic sequences is not entirely clear. However, what are some potential functions? - ANSWERS: - Some DNA regions directly participate in the regulation of gene expression (promoters, termination signals, etc.)

• Some DNA encodes for small regulatory RNA with poorly understood functions
• Some DNA may be junk (pieces of unwanted genes, remnants of viral infections) 50% of the human genome is repetitive, made up of many simple repeat sequences (_______, satellites), especially at _______________ and telomeres. - ANSWERS: STRs; centromeres __________________ can move around within the genome. - ANSWERS: Transposons Note: Millions of copies of the Alu element, 11% of genome, 80-100 still mobile and can disrupt genes

Determine if the following describes a centromere or telomere: A. TTAGGG in humans (1500 repeats) B. Essential for equal distribution of chromosomes C. Attachment point for kinetochore proteins and microtubules D. End of chromosomes E. Central region of chromosomes F. 130 bp A-T rich repeat G. Shortened each round of replication up to Hayflick limit (52 divisions) H. Where the two daughter chromosomes are held together during mitosis - ANSWERS: A. Telomere B. Centromere C. Centromere D. Telomere E. Centromere F. Centromere G. Telomere H. Centromere DNA in the cell must be organized to allow ____________ of large DNA molecules within the cells, and access for ____________ to read the information in the DNA sequence. - ANSWERS: Packing; proteins There are several levels of organization, one of which is the ___________________ of the double- stranded DNA helix. - ANSWERS: Supercoiling What is non-supercoiled DNA also called? - ANSWERS: Relaxed DNA Supercoiling has great influence on _________________ and replication of DNA, and highly regulated. - ANSWERS: Transcription

Plectonemic is seen in ______________, and has a _______-handed superhelix. - ANSWERS: Plasmids; right Toroid/solenoid is found in _____________, and has ______-handed turns. - ANSWERS: Chromatin; left Note: It is very compact, and resembles a garden hose on a reel Normal B-form DNA is ______________ and has _____ bp/turn, but most cellular DNA is _________________. - ANSWERS: Relaxed; 10.5; underwound True or false: Circular DNA is relaxed. - ANSWERS: False; Circular DNA is rarely relaxed, as the fewer helical turns (underwound) makes strand separation easier. This strain induces supercoiling. Linear DNA is ________________ with the help of proteins to prevent strands from _______________. - ANSWERS: Underwound; rotating True or false: Twist is the number of supercoils. - ANSWERS: False; Twist is the number of twists and tuns of the two strands of the double helix. Writhe is the number of supercoils (typically negative). In circular and long linear DNA, changing twist and writhe requires _____________ a strand. - ANSWERS: Breaking What is the linking number? - ANSWERS: Tw + Wr; the number of times the DNA strands wind around one another in total, including both twist and writhe Topoisomerases change ___________ ____________, and are required for DNA unwinding and rewinding during transcription and _________________. - ANSWERS: Linking number; replication

True or false: Type I topoisomerases remove negative supercoils and relax DNA via single-stranded breaks, and change linking number by +/1 (twist). - ANSWERS: True Note: Type II topoisomerases relax supercoils in humans via double-stranded breaks, use ATP, change linking number by +/- 2 (writhe). True or false: DNA gyrase in E. coli can introduce supercoils. - ANSWERS: True Topoisomerases use an active site __________ to facilitate the reaction, which binds _______________ to the DNA backbone. - ANSWERS: Tyrosine; covalently Prokaryotes use __________ to introduce negative supercoiling, and targeted by _______________________. - ANSWERS: Gyrase; fluoroquinones In eukaryotes, ____________ cells are susceptible to topoisomerase inhibitors like topotecan, camptothecin, and doxorubicin. - ANSWERS: Cncer What may also inhibit topoisomerase II? - ANSWERS: Reseveratrol and other natural polyphenols Cells have a ________________ state called G0, interphase with _______, Synthesis (S), and Gap2 (G2), where chromatin is _______________ and randomly dispersed. - ANSWERS: Nondividing; Gap1; undefined True or false: During anaphase of mitosis, chromosomes become condensed, and pairs of sister chromatids form. - ANSWERS: False; This occurs during prophase of mitosis. True or false: 2 meters of DNA fits into a 5-10 micrometer nucleus. - ANSWERS: True DNA associates tightly with proteins called ____________, which are small and very basic, 25% _____ and _______. - ANSWERS: Histones; Lys and Arg

Note: Histones can be modified by methylation, acetylation, phosphorylation, glycosylation, SUMOylation, and ubiquitination. 147 bp of DNA wraps around histone octamers called __________________. - ANSWERS: Nucleosomes Nucleosomes link up to form ________________, which consists of fibers of DNA and proteins, with some RNA. - ANSWERS: Chromatin True or false: Histone binding is random. - ANSWERS: False; Histone binding is not random, and it occurs more often at AT/GC repeats. What does wrapping DNA around the histone core require? - ANSWERS: Removal of one helical turn The underwinding occurs without a strand break. so a compensatory positive supercoil forms, which is relaxed by topoisomerase. This leaves DNA with a ___________ ________________, forming a left- handed solenoid. - ANSWERS: Negative supercoil Nucleosome formation compacts DNA 7x, but overall compaction is >10,000x. __ binds to the DNA to create the 30 nm fiber. - ANSWERS: H What are the two models for nucleosome formation? - ANSWERS: Solenoid model (humans) and the zigzag model True or false: Higher-order structures are not well understood, and may vary between chromosomes, regions, and time. - ANSWERS: True Higher-order structures appear to involve a _____ of DNA associating with a ______________ of proteins such as topoisomerase and SMCs. - ANSWERS: Loop; scaffold

Condensed chromosome structure is maintained by _____ ___________, which are rings with ATP- binding sites. - ANSWERS: SMC proteins True or false: Cohesins wraps around a single chromatid and condense it. - ANSWERS: False; Cohesins link two sister chromatids and hold the chromosomes together in mitosis (cleaved by separase). Condensins wrap around a single chromatid and condense it. Cohesins are added during ___ _________ and removed during _____________. - ANSWERS: S phase; anaphase Condensins are added during ________________, and removed during ________________. - ANSWERS: Prophase; telophase DNA provides for the stable storage of ______________ information, and it is a _________________ structure. - ANSWERS: Genetic; dynamic A new copy of DNA is synthesized with ______ ____________ before each cell division, errors during synthesis must be ________________, and segments of DNA can rearrange via ____________________.

• ANSWERS: High fidelity; repaired; recombination Who noted a possible copying mechanism in their 1953 paper? - ANSWERS: Watson and Crick What did the Meselson-Stahl experiment do? - ANSWERS: Prove DNA replication was semiconservative In the Meselson-Stahl experiment, Cells were grown on a medium containing only ______ nitrogen until all the DNA became fully labeled, which produced one band when centrifuged in _______.

Cells were then switched to _______ nitrogen and allowed to divide once, which produced a ________ band of ________________________________________. Continued division showed even a _________ band which contained _________________________. - ANSWERS: Heavy; CsCl; Light; medium; dsDNA containing both heavy and light nitrogen; Higher; only light nitrogen True or false: Synthesis always occurs 3' -> 5' by addition of new nucleotides to the 5' end. - ANSWERS: False; Synthesis always occurs 5' -> 3' by addition of new nucleotides to the 3' end (3'- OH) True or false: The lagging strand is made discontinuously in short pieces (Okazaki fragments) that are ligated. - ANSWERS: True Note: The leading strand is made continuously as the replication fork advances. True or false: Replication is made unidirectionally in circular DNA. - ANSWERS: False; Replication is bidirectional in circular DNA, still with two leading/lagging strands. DNA polymerase is a transferase that has three domains. Match these domains to the correct functions: A. Palm B. Fingers C. Thumb

1. Catalytic site, with a pre/postinsertion site

2. Bring the incoming bases into the active site
3. Holds polymerase to the DNA - ANSWERS: A- 1 B- 2 C- 3 Polymerases have widely varying ________________, which is the amount of DNA they can copy before falling off. - ANSWERS: Processivity When elongating, the parental DNA strand serves as a ______________. - ANSWERS: Template ________________________ ____________________ serve as substrates in strand synthesis, which incorporates a nucleotide. - ANSWERS: Deoxyribonucleotide triphosphates (dNTPs) The nucleophilic 3'-OH group of the growing chain attacks the ________________ of the incoming trinucleotide, which is facilitated by nearby _____ ions. - ANSWERS: α-phosphate; Mg2+ ________________________ is a good leaving group, and is further cleaved to pull the reaction forward.

• ANSWERS: Pyrophosphate What is pyrophosphate made of? - ANSWERS: The β and γ phosphates True or false: Base pairing ensures high fidelity. - ANSWERS: True DNA polymerase can't detect the base, but the __________________ of the base pair. - ANSWERS: Geometry Note: The DNA polymerase active site excludes base pairs with incorrect geometry. DNA polymerases still insert wrong base 1/1000 - 1/10000 times, but repair mechanisms fix errors. Another note: The error rate in E. coli is 1/10^9 -1/10^10 bp (1 per 1000-10000 replications)

True or false: Most DNA polymerases have additional proofreading activity. - ANSWERS: True True or false: DNA polymerase's 5' -> 3' exonuclease activity "proofreads" synthesis for mismatched base pairs. - ANSWERS: False; DNA's polymerase's 3' -> 5' exonuclease activity "proofreads" synthesis for mismatched base pairs. Note: Synthesis will stall until the incorrect nucleotide is removed. True or false: DNA polymerase I is abundant and is ideal for replication. - ANSWERS: False; DNA polymerase I is abundant (400 per cell) but it is not ideal for replication. Note: Its rate (600 nucleotides/min) is slower than the replication fork movement. Another note: It has low processivity, and its primary function is in clean-up with its exonucleases (3' -> 5' proofreading, 5' -> 3' base removal) What role does DNA polymerase III have? - ANSWERS: It is the principal replication polymerase Which DNA polymerases are involved in DNA repair? - ANSWERS: DNA polymerases II, IV, and V DNA polymerase III is a ________________ with 10 subunits. - ANSWERS: Holoenzyme In DNA polymerase III, two core domains are linked by the clamp loader complex's _____ ____________.

• ANSWERS: Tau protein In DNA polymerase III, the core domains interact with a dimer of ___ _______________ ____________ that prevent dissociation and increase the processivity to >500,00 bp. - ANSWERS: β sliding clamps

Note: E. coli replication requires over 20 proteins, which is together called the replisome (polymerases, helicases, topoisomerases, DNA-binding proteins, primases, ligases, etc.) ________________ (245 bp) is AT-rich, which is recognized and bound by DnaA. - ANSWERS: E. coli oriC _________ is the initiator, which is an ATPase that bends the DNA. - ANSWERS: DnaA _____ __________ is a hexamer that is opened by the binding of DnaC, which unwinds the DNA strands.

• ANSWERS: DnaB helicase True or false: DNA polymerase III links to DnaB. - ANSWERS: True ______________________________________ stabilizes separated strands. - ANSWERS: Single-stranded DNA-binding proteins (SSBs) ________________________ relieves topological stress ahead of the replication forks. - ANSWERS: Topoisomerase True or false: Replication must only occur once per cycle. - ANSWERS: True DnaA's ATP is ________________, and it dissociates and reassociates 20-40 minutes later. - ANSWERS: Hydrolyzed After replication, oriC is _________________ by Dam methylase at _________, which recruits _________. - ANSWERS: Hemimethylated; GATC; SeqA Note: After a period, SeqA dissociates, and the oriC is available to be fully methylated, allowing for another round of replication. Primase interacts with _______________ and makes an RNA primer, which is then handed off to the clamp loader (which adds the sliding clamp) and then DNA polymerase III. - ANSWERS: Helicase