Genetics Unit 2 Chapters 6 and 7
key features of DNA replication that reflect 3 strict requirements for DNA polymerase action
1. the four dNTPs 2. a single-stranded template. Double-stranded DNA must be unwound, and DNA polymerase moves along the template in the 3' to 5' direction 3. A primer with a free 3' hydroxyl group. DNA polymerase cannot establish the first link in the chain, and the enzyme adds nucleotides successively to the 3' end of the growing DNA chain. (that is, DNA polymerase synthesizes DNA only in the 5' to 3' direction)
Benzer used recombination frequencies to produce a map of the "fine structure" of the rII region. What's the formula?
RF (in map units) is the number of rII+ recombinants divided by the total number of phages, multiplied by 2 to account for the rII- double mutant reciprocal recombinants that must exist but cannot be easily detected
prototroph
a cell that does not require the addition of a substance. refers to a wild-type cell that can grow on a minimal medium alone
point mutation
a change in a single base pair in the genetic material
complementation group
a collection of mutations that do not complement each other. often used as a synonym for "gene" bc the mutations in a complementation group all affect the same unit of function, and thus, the same gene
gene
a functional unit that directs the appearance of a molecular product that, in turn, contributes to a particular phenotype typical size range is 1,000-20,000 bp
missense mutation
a genetic alteration that causes the substitution of one amino acid for another
DNA topoisomerases
a group of enzymes that helps relax the supercoils by nicking one or cutting both strands of DNA--that is, cleaving the sugar-phosphate backbone between two adjoining nucleotides.
complementary base pairing
a process during DNA replication where newly replicated strands form as complementary bases align opposite the exposed bases on the two parental strands. an A at one position on the original strand signals the addition of a T at the corresponding position on the newly forming strand
transversion
a type of base substitution that involves a change of a pyrimidine to a purine, or vice versa
transition
a type of base substitution that involves a change of a pyrimidine to another pyrimidine, or a purine to another purine.
exonuclease
an enzyme that removes nucleotides from an end of a DNA molecule the 3' to 5' exonuclease activity of the enzyme allows it to be excised. known as proofreading
resolvase
an enzyme that separates the two interlocked nonsister chromatids by the breakage of two DNA strands at each Holliday junction. because it almost always cuts all four DNA strands, resolution of the double Holliday junctions usually results in crossing over
thymine-thymine dimers
assault of ultraviolet light, which causes adjacent thymine residues to become chemically linked into these
homologous recombination
cells can use this process to repair most double-stranded breaks accurately through complementary base pairing.
amino acids
cells use mainly 20 different amino acids to synthesize the proteins they need. basic features are encapsulated by the formula NH2-CHR-COOH. The -COOH component (carboxylic acid) is acidic; the -NH2 component (amino group) is basic. R refers to the side chains that distinguish each of the amino acids.
chromosome mutations
changes in the number or structure of chromosomes
tautomers
each of the four bases has two of theses similar chemical forms that interconvert continually
polymerase I
fills in the gaps between newly synthesized Okazaki fragments
somatic mutations
in cells that do not produce gametes
germ line mutations
in cells that produce gametes a mutation that occurs directly in a sperm or egg cell, or in one of their precursor cells. mutations that arise in these cells are the only novel mutations that can be passed down to progeny
secondary structure
localized regions with characteristic geometry. determined by the sequence of amino acids in the polypeptide
reverse mutation (reversion)
mutations that cause a novel mutant allele to revert back to wild type. wild type alleles are designated with a (+), whether recessive or dominant to mutant allele
polymerase III
plays the major role in producing the new strands of complementary DNA
2 stages of DNA replication: elongation
proteins connect the correct sequence of nucleotides on both newly formed DNA double helixes
anticrossover helicase
recombination initiated by Spo11 can also result in no crossing over though the action of this enzyme. The helicase helps disentangle the invading strand from the nonsister chromatid, thus interrupting Holliday junction formation. although the end result is no crossing over, one of the resultant chromatids nonetheless contains a heteroduplex region
oligopeptide
several amino acids linked together by peptide bonds
Luris and Delbruck's fluctuation test
showed that mutations in bacteria conferring resistance to bacteriophage occur prior to exposure to the phages and are cased by random, spontaneous events
mutation hotspots
sites within a gene that spontaneously mutate more frequently than others. their existence suggests that certain nucleotides can be altered more readily than others.
primary structure
the linear sequence of amino acids within a polypeptide. each unique primary structure places constraints on how a chain can arrange itself in 3D space. the primary structure determines 3D shape by generating secondary structure. also responsible for other folds and twists that together with the secondary structure produce the ultimate 3D tertiary structure of the entire polypeptide. no other info beyond the primary structure is needed to achieve the proper 3D shape of such proteins
Steps for DNA replication: Elongation
-Connecting the correct sequence of nucleotides into a continuous new strand of DNA. -DNA pol III catalyzes the joining of a new nucleotide to the preceding nucleotide. the linkage of subunits through the formation of phosphodiester bonds is known as polymerization. DNA pol III first joins the correctly paired nucleotide to the 3' hydroxyl end of the RNA primer, and then it continues to add the appropriate nucleotides to the 3' end of the growing chain. DNA strand grows in the 5' to 3' direction and DNA pol molecule moves along the antiparallel template in the 3' to 5' direction. helicase progressively unwinds the double helix. DNA pol III moves in the same direction as the fork to synthesize the leading strand. the lagging strand is synthesized discontinuously in the normal 5' to 3' direction as small fragments of about 1000 bases called Okazaki fragments. Finally, DNA pol I and other enzymes replace the RNA primer of the previously made Okazaki fragment with DNA, and an enzyme known as DNA ligase covalently joins successive Okazaki fragments into a continuous strand of DNA
replica plating
-a technique to demonstrate that the mutations conferring bacterial resistance occur before the cells encounter the bactericide that selects for their resistance. -a technique in which bacterial colonies are transferred from one plate to another with a sterile piece of velvet cloth
genes encode...
-not all genes govern the construction of enzymes active in biochemical pathways. enzymes are only one class of the molecules known as proteins, and cells contain many other kinds of proteins. -among the other types are proteins that provide shape and rigidity to a cell -proteins that transport molecules in and out cells -proteins that help fold DNA into chromosomes -proteins that act as hormonal messengers -genes determine the synthesis of all proteins, enzymes, and nonenzymes alike -genes determine the construction of polypeptides, and bc some proteins are composed of more than one type of polypeptide, more than one gene determines the construction of such proteins
Steps for DNA replication: Initiation
-preparing the double helix for use as a template -initiation begins with the unwinding of the double helix at a particular short sequence of nucleotides known as the origin of replication. Several proteins bind to the origin, starting with the initiator protein. Initiator attracts DNA helicase, which unwinds the double helix. the opening up of a region of DNA generates two Y shaped areas called replication forks, one at either end of the unwound area, the replication bubble. The single strands will serve as templates for synthesizing new strands of DNA. An enzyme complex known as DNA polymerase III adds nucleotides to the 3' end of a preexisting strand of nucleic acid. the requirement for an already existing chain means that something else must prime the about-to-be-constructed chain. that "something else" is a short stretch of RNA called an RNA primer, synthesized by an enzyme called primase
base excision repair
-removes damaged bases -glycosylase enzymes remove aberrant bases, leaving an AP site. AP endonuclease cuts the sugar-phosphate backbone, making a nick at the AP site. -DNA exonucleases remove nucleotides near the nick, creating a gap -DNA polymerase synthesizes new DNA to fill in the gap -DNA ligase seals the nick
gene conversion
-the physical change of one allele in a heterozygote into the other. -sometimes occurs as a result of a recombination event
Steps in experiment done by Hershey and Chase where they showed that DNA is the genetic material of bacteriophage T2
1. Incubated 35S-labeled protein bacteriophage or 32P-labeled DNA bacteriophage with E. coli in separate flasks 2. allowed time for infection of bacteria by virus 3. sheared virus coats from cell walls of bacteria using a blender 4. Centrifuged the blended E. coli/virus mixture 5. measured radioactivity in the supernatant of the centrifuged material using a Geiger counter after centrifugation, no sulfur in cells, but there is phosphorous. Shows DNA is the carrier of information bc it was injected inside the cell to infect it.
differences between RNA and DNA
1. RNA takes its name from the sugar ribose, which it incorporates instead of the deoxyribose found in DNA. 2. RNA contains the base Uracil (U) instead of the base Thymine (T). U pairs with A 3. most RNA molecules are single stranded and contain far fewer nucleotides than the very long DNA molecules in nuclear chromosomes.
Benzer's 3 conclusions about gene structure and function based on his observations with the rII genes
1. a gene consists of different parts that can each mutate 2. recombination can occur between different mutable sites in the same gene 3. a gene performs its normal function only if all of its components are wild type
order of events of the replicative cycle of bacteriophage T2
1. attachment of virus to cell wall of bacterium by tail fibers of virus 2. injection of viral genetic material into cytoplasm of bacterial cell 3. production of viral proteins and genomes 4. viral assembly 5. release of virus by lysis of host bacterium
Steps for a meiotic recombination event
1. begins when Spo11 makes a double-strand break in one of the four chromatids 2. an exonuclease degrades one strand of DNA from both sides of the cleavage, leaving 3' single-stranded tails 3. in the next set of reactions, called strand invasion, one single-stranded tail displaces the corresponding strand on the non sister chromatid 4. Strand invasion results in the formation of a heteroduplex region in which the DNA molecule is composed of one strand from each non sister chromatid. 5. the strand displaced by strand invasion forms a second heteroduplex with the other 3' single-stranded tail. 6. DNA synthesis to extend the two 3' tails replaces the DNA that was degraded by the exonuclease, and DNA ligase reseals the DNA backbones 7. The result is that the two non sister chromatids are interlocked at two Holliday junctions. the Holliday junctions move away from each other and thereby enlarge the heteroduplex between them--a process called branch migration. now, the two non sister chromatids must be separated.
forward mutation
a mutation that changes a wild-type allele of a gene to a different allele. the resulting allele can be either recessive or dominant to the original wild-type allele. diagramed as A -> a when the gene is recessive to the wild type and as b -> B when the mutation is dominant to the wild type.
auxotroph
a nutritional mutant microorganism that requires supplementation with substances not needed by wild-type strains
dipeptide
a pair of amino acids connected by a peptide bond
Spo11
a protein that plays crucial roles in initiating meiotic recombination in yeast, and is homologous to a protein essential for meiotic recombination in nematodes, plats, fruit flies, and mammals. finding suggests that the mechanism of recombination known as the "double-strand-break repair model" has been conserved throughout the evolution of eukaryotes
hydroxylamine
adds -OH to cytosine, allowing it to pair with adenine
ethylmethane sulfonate
adds an ethyl group to guanine or thymine, which then pair incorrectly
the two kinds of chemical groups that represent the ends of a polypeptide
amino and carboxyl
resolution
breakage and rejoining of DNA strands to produce separate chromosomes at the end of recombination
selection
conditions are such that the only survivors are the rare individuals you seek to identify
DNA ligase
during elongation, this enzyme welds together Okazaki fragments
peptide bonds
during protein synthesis, a cell's protein-building machinery links amino acids by constructing covalent ___ that join the -COOH group of one amino acid to the -NH@ group of the next.
redundancy
either strand of the double helix can specify the sequence of the other. This redundancy provides a basis for checking and repairing errors arising from either chemical alterations sustained during storage or from rare malfunctions of the replication machinery
telomeres
eukaryotic chromosomes have evolved specialized termination structures known as ___, which ensure the maintenance and accurate replication of the two strands of each linear chromosome
the remarkable precision of the cellular replication machinery
evolution has perfected the cellular machinery fro DNA replication to the point where errors during copying are exceedingly rare.
primase
generates RNA primers to initiate DNA synthesis
template
in the process of replication, the double helix unwinds to expose the bases in each strand of DNA. each of the two separated strands then acts as a _____, or molecular mold, for the synthesis of a new second strand.
mutation
inherited change in the genetic information (heritable change in the DNA sequence)
proflavin
intercalates into the double helix, causing deletions and insertions
5-Bromo-uracil mutagen
is incorporated into DNA instead of thymine, but can pair with guanine
single stranded DNA binding proteins
keep the DNA helix open
native configuration
normal tertiary structure--the way a long chain of amino acids naturally folds in 3D space under physiological conditions. various forces, including hydrogen bonds, electrostatic bonds, hydrophobic interactions and disulfide bridges help stabilize the native configuration
substitution
occurs when a base at a certain position in one strand of DNA molecule is replaced by one of the other three bases. can be divided into transitions, in which one purine (A or G) replaces the other purine, or one pyrimidine (C or T) replaces the other; and transversions, in which purine changes to a pyrimidine, or vice versa
deletion
occurs when a block of one or more nucleotide pairs is lost from a DNA molecule
proteins
polymers composed of building blocks known as amino acids
N terminus
polypeptides have a chemical polarity one end is called this because it has a free amino group that is not connected to any other amino acid
recombinants
products of crossing over chromosomes that carry a mix of alleles derived from different homologs
in which locations can circular chromosomes be found
prokaryotic, chloroplasts, mitochondria, papovaviruses
3 ways that organisms ensure the information fidelity of its DNA
redundancy the remarkable precision of the cellular replication machinery enzymes that repair chemical damage to DNA
nucleotide excision repair system
removes alterations that base excision repair cannot repair because the cell lacks a DNA glycosylase that recognizes the problem base. main system used in the repair of bulky, helix distorting lesions -a complex of the UvrA and UvrB proteins patrols the DNA for irregularities, detecting lesions that disrupt base pairing and thus distort the double helix (such as thymine-thymine dimers). At the damaged site, UvrA dissociates with UvrB, allowing UvrB to associate with UvrC. these enzymes nick the DNA exactly 4 nucleotides to one side of the damage and 7 nucleotides to the other side, releasing a small fragment of single-stranded DNA. DNA polymerases then resynthesize the missing information and DNA ligase reseals the now-corrected strand.
nitrous acid
removes an amine group (deaminates) from adenine or cytosine, changing their pairing properties. leads to mutations in the newly-synthesized DNA strand during DNA replication
methyl-directed mismatch repair
the 100-fold higher accuracy of the cell depends on this backup system that notices and corrects residual errors in the newly replicated DNA. -in bacteria, this mechanism corrects mistakes in replication -parental strands are marked with methyl groups -MutS and MutL recognize mismatch in replicated DNA -MutL recruits MutH to GATC; MutH makes a nick in strand opposite methyl tag (newly synthesized strand) -DNA exonucleases excise DNA from unmethylated new strand -repair and methylation of newly synthesized DNA strand
quartenary structure
the 3D configuration of subunits in a multimer. the same forces that stabilize the native form of a polypeptide also contribute to the maintenance of quartenary structure. determined by interactions between polypeptides
insertion
the addition of one or more nucleotide pairs
supercoiling
the additional twisting of the DNA molecule. movement of the replication fork causes more of this
polypeptides
the amino acid chains that make up proteins and contain hundreds to thousands of amino acids joined by peptide bonds
replication forks
the antiparallel strands of DNA unwind gradually at the two Y shaped areas called the _____
enzymes that repair damage to DNA
the cell has an array of enzymes devoted to the repair of nearly every imaginable type of chemical damage
multimer
the complex of subunits. multimeric proteins include two or more polypeptides (subunits). if these subunits are different, they must be encoded by different genes
photolyase
the enzyme that can recognize thymine dimers and split them. the photolyase enzyme works only in the presence of visible light, so in carrying out its DNA repair tasks, it associates with a small molecule called a chromophore that absorbs light in the visible range of the spectrum; the enzyme then uses the energy captured by the chromophore to split thymine-thymine dimers. bc it does not function in the dark, the photolyase mechanism is called light repair or photorepair.
tautomerization
the equilibrium between the tautomers is such that each base is almost always in the form in which A pairs with T and G pairs with C. However, if by chance a base in the template strand is in its rare tautomeric form when DNA polymerase arrives, the wrong base will be incorporated into the newly synthesized chain because the rare tautomers pair differently than do the normal forms. -rare form of C pairs with A -rare form of G pairs with T -rare form of A pairs with C -rare form of T pairs with G
recombination
the generation of new allelic combinations through genetic exchange between homologous chromosomes
subunits
the individual polypeptides in an aggregate
depurination
the kind of DNA alteration where the hydrolysis of a purine base, A or G, from the deoxyribose-phosphate backbone occurs 1000 times an hour in every human cell. because the resulting apurinic site cannot specify a complementary base, the DNA replication process introduces a random base opposite the apurinic site, causing a mutation in the newly synthesized complementary strand three quarters of the time
Okazaki fragments
the lagging strand is made at the replication fork and is synthesized by the other template (the one not synthesizing the leading strand). It is generated in pieces called ___ as more and more template is unwound at the fork. fragments are then joined together
X ray diffraction
the main technological development that allowed Crick and Watson to work out the structure of the DNA double-helix
biochemical pathway
the orderly series of reactions that allows enzymes to obtain simple molecules from the environment and convert them step-by-step into successively more complicated molecules culminating in an amino acid
C terminus
the other end of the polypeptide chain that contains a free carboxy group
semiconservative replication
the pattern of double helix duplication: a copying in which one strand of each new double helix is conserved from the parent molecule and the other is newly synthesized
2 stages of DNA replication: initiation
the proteins open up the double helix and prepare it for complementary base pairing
deamination
the removal of an amino (-NH2) group. can change cytosine to uracil, the nitrogenous base found in RNA but not DNA. Uracil base pairs with A now, instead of C base pairing with G.
tertiary structure
the ultimate 3D structure of the entire polypeptide determined by the sequence of amino acids in the polypeptide
What best summarizes Garrod's hypothesis about alkaptonuria and other "inborn errors of metabolism"?
they arise from a heritable mutation that creates a damaged enzyme for a specific biochemical reaction
nonhomologous end joining
this mechanism is especially important for the repair of double-stranded breaks formed during the G1 phase of the cell cycle (before a sister chromatid is available to serve as a template for homologous recombination -double strand break in the DNA -the proteins participating in NHEJ bind to DNA ends at the site of the breakage and protect the ends from nucleases. the NHEJ proteins also bridge the two ends -end joining by DNA ligase.
3' to 5' exonuclease
this nuclease portion of the polymerase molecule recognizes a misfired base and excises it, allowing the polymerase to copy the nucleotide correctly on the next try. this editing function improves the fidelity of replication a 100-fold
what was the utility of the deletion mapping approach used by Benzer?
to quickly map point mutations to a region
point (gene) mutations
transitions, transversions, small deletions, and insertions that affect one or just a few base pairs in the DNA and thus alter only one gene at a time
DNA helicase
unwinds the double helix
cistron
what Benzer called any complementation group identified by the cis-trans test. test includes the complementation test and the control experiment. in the complete experiment, two mutations that do not produce lysis in trans but they do in cis are in the same complementation group
denatured
when many proteins unfold when exposed to urea and mercaptoethanol or to increasing heat or pH