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Genetics Exam 3
Terms in this set (70)
DnaA protein function?
binds to DnaA boxes with origin to initiate DNA replication.
Function of DnaB protein?
AKA DNA helicase, separates double-stranded DNA.
Function of DnaC protein?
Aids DnaA in recruitment of DNA helicase to origin.
Function of (bacterial) Topoisomerase?
Removes positive supercoiling ahead of replication fork
Function of (bacterial) Primase?
Synthesizes short RNA primers
Function of DNA (bacterial) polymerase III?
Synthesized DNA in leading and lagging strands.
Function of DNA (bacterial) polymerase I?
Removes RNA primers, fils in gaps with DNA.
Function of SSB protein?
Binds to single-stranded DNA and prevent from reforming double-stranded structure.
Function of DNA ligase?
Covalentyl attaches adjacent Okazaki fragments.
Function of Tus protein?
Binds to ter sequences preventing advancement of replication fork (terminates replication)
Components of oriC.
AT-rich region, DnaA boxes, GATC methylation sites.
Function AT-rich regions
relatively easy to melt into single strands, are required for the initial opening of the double helix.
Function of DnaA boxes
DNA sequences in oriC that are required to bind DnaA protein. DnaA protein
binds in complex to open the double helix and initiate replication.
Function alpha, epsilon, theta, beta-subunits of Prokaryotic DNA Polymerase III holoenzyme?
a: DNA synthesis
epsilon: 3'-5' proofreading
theta: accessory protein stimulates proofreading function
beta: clamp protein allowing polymerase to slide along DNA w/o falling off.
What aids the processive feature of Polymerase III?
B - clamp protein
y (gamma) - clamp loader protein (initial clamp)
What are the causes behind DNA replication fidelity?
Instability of mismatched pairs, proofreading functions, configuration of DNA polymerase active site.
How does proofreading happen?
Polymerases use their 3'-5' exonuclease activities to remove incorrect nucleotides.
Describe mechanisms of initiation regulation
• Changing conformation of DnaA protein to inactive, reducing relative concentration
of active DnaA protein to DnaA boxes.
• Methylation of GATC sequences at oriC by Dam methylase.
What is the enzyme responsible for methylation of GATC sites in oriC?
DNA adenine methytransferase methylase (Dam methylase)
What are ARS elements?
Autonomously Replicating Sequences. Relate to origins of replication sites for EUKARYOTES. Contain high A-T percentages. 3-4 copies of a specific sequences.
What is the function of a, epsilon, delta, gamma types eukaryotic polymerases?
a: initiates DNA replication in conjunction w/ primase.
epsilon: replication of leading strand during s-phase
delta: replication of lagging strand
gamma: replication of mitochondrial DNA.
What removes the RNA primer in eukaryotic DNA replication?
NOT DNA polymerase. Special flap endonuclease does this.
What does a Telomeric sequence consist of?
Ends of linear eukaryotic chromosomes. 3' overhang 12-16 nucleotides long, some guanine and many thymines. Humans: TTAGGG.
What is the function of telomerase?
Contains complementary RNA that binds to 3' overhand acts as a template for the extension of DNA by reverse transcriptase. Purpose is to maintain telomere length after each cycle of DNA replication.
If the repeat structure of a telemore at right overhang end of chromosome is 5-TTAGGG-3, what is it on the left underhang?
Describe mechanism of processivity in DNA replication
Beta clamp protein keeps DNA Pol III bound to the template DNA during synthesis, maintaining its high speed speed by preventing the DNA Pol III from falling off.
What are the functions of the RNA and protein components of telomerase enzyme?
the RNA component binds to the telomere DNA so that the protein component can synthesize repeats (reverse transcriptase), using the RNA component as a template
What is a point mutation?
A change in a single base pair. Involves base substitution.
Distinguish between transition and transversion
A transition is a change of a pyrimidine to another
pyrimidine or a purine to another purine.
A transversion is a change of a pyrimidine to a purine or
What is a silent mutation?
base substitutions that do not
alter the amino acid sequence of the polypeptide (protein is the same)
What is a missense mutation?
base substitutions in which
an amino acid change does occur. Protein may or may not (neutral mutation) be affected.
What is a nonsense mutation?
base substitutions that
change a normal codon to a termination/stop codon.
What is a frameshift mutation?
involve the addition or deletion of
nucleotides in multiples of one or two. Completely different AA sequence occurs downstream from mutation.
What are some examples of gene sequence mutations outside the coding sequence?
Promoter (rate of transcription), 5-UTR/3-UTR (ability/stability of mRNA to be translated), splice recognition sequences (proper splicing of pre-mRNA)
What is a null mutation?
Removal of all functions of a gene, gives the opposite/recessive corresponding phenotype.
What experiment proved random mutation theory?
Lederbergs' Replica Plating experiment.
Distinguish between spontaneous and induced mutations.
- Result from abnormalities in cellular/biological processes
- Caused by environmental agents
Describe Depurination mutation
Breaking of the link between purines and deoxyribose. Base is lost.
Describe Deamination mutation
Cytosine and 5-methylcytosine can deaminate to create Uracil or Thymine e.g. C--> U/T (mispairing, similar to base modifiers)
Give examples of spontaneous and induced mutation.
• Transposable elements, depurination, deamination, tautomeric shifts.
• Chemical, physical (UV, Xrays) agents.
Describe tautomeric shift mutations
Conversion of bases into enol/imino forms. Causes a promotion of mispairing (i.e. AC, GT pairs)
Describe Oxidation of Bases by reactive oxygen species (ROS) mutations
Oxidizes Guanine bases that promotes binding with Adenine (transition)
What are the three types of chemical mutagens?
• Base modifers: change base pairing
• Intercalating agents: distort, cause single base deletions/additions
• Base analogies: undergo tautomeric shifts more frequently (Pur-Pyr mispairing)
What chemical mutagens can cause mispairing mutations?
Base analogues, base modifiers.
What chemical mutagens can cause additions/deletions?
What are two types of physical mutagens?
Ionizing (Xrays, Gamma rays, penetrating, create free radicals), non-ionizing radiation (UV, nonpenetrating, creates thymine dimers)
What is the effects of Ionizing radiation?
Base deletions, single nicks, cross-linking, chromosomal breaks
What is the effects of non-ionizing radiation?
Creates of thymine dimers
What test is used for determining if an agent is a mutagen? Describe.
Ames test. Monitors rate of a second mutation (reversion) in Salmonella typhimurium involving a histidine synthesis gene.
Describe Ame's test in detail. Significance?
Create tube 1 (control) with rat liver. Create tube 2 with rat liver, salmonella strain (his-), and mutagen. Plate mixtures and incubate. Larger number of colonies suggest mutagen causes mutation into salmonella(his+).
Explanation: Mutagen causes point mutation that allows salmonella to synthesize histidine increasing colony growth.
Describe Replica plating in detail. Significance?
Grow plate of T1phage-absent colonies. Press cloth on plate to create replica of plate colonies. Press cloth onto 2 copies of T1page plate. Result: identical colony locations on T1 secondary plates, selected for previously occurring mutants.
What is the role of Photolyase in reversal of mutant covalent modifications?
Directly repairs thymine dimers
What is the role of O6-alkyguanine alkyltransferase in reversal of mutant covalent modifications?
Directly repairs alkylated bases by transferring the methyl/ethyl group from base to a cysteine side
What is the role of DNA N-glycosylase?
Involved in Base Excision Repair. Recognizes abnormal base and cleaves bond between it and sugar. Eliminates abnormal uracil, thymine dimers.
What two molecules/systems can eliminate thymine dimers?
Photolyase, DNA N-glycosylase (BER), Nucleotide excision repair (NER)
Describe the mechanism of Prokaryotic Nucleotide Excision Repair
• UvrA/UvrB tracks along DNA, detects damage.
• UvrA releases.
• UvrC binds and cuts both sides of damage.
• UvrD (helocase) removes damaged region.
• UvrB/UvrC releases.
• DNA polymerase fills gap.
• DNA ligase seals gap.
Describe mechanism of prokaryotic Mismatch Repair System
• MutS of the MutS/MutL complex finds mismatch.
• MutS/MutL complex binds to MutH which is prebound to hemimethylated sequence.
• MutH cutes NON-METHYLATED strand.
• MutU segregates DNA strands.
• Exonuclease digests nonmethylated strand beyond mismatch.
• DNA Polymerase fills.
• DNA ligase seals.
What is the importance of of dam-mutant organism?
The absence of dam-methylase (mutation) results in the absence of hemimethylated DNA strands making DNA repair mechanism 50% more likely to fails due to the inability to distinguish between strands.
What are inverted repeats?
DNA sequences at each end of element that are identical but on opposite DNA strands.
What are direct repeats?
DNA sequences adjacent to IR but not considered a part of the element. Made after element inserts.
Functions of retroviruses?
RNA virus that makes DNA copy by reverse transcriptase. DNA integrates into host genome by integrase. Integrated DNA, using promoter in Long Terminal Repeat, transcribes viral RNA.
Describe mechanism of cut-and-paste transposition
Transposase protein binds inverted repeats and cuts double helix beside them. Transposase nicks target site base pairs and inserts (TE) DNA element. Gaps are filled by DNA polymerases, nicks sealed by ligase.
What gene codes for reverse transcriptase and integrase?
What is Ectopic homologous recombination?
Recombination between transposable elements at different site in a genome. Possible because element sequences are the same.
What are the two explanations of the existence of transposons?
• Selfish DNA theory - act like parasites
• Exon shuffling, off evolutionary advantage (i.e. carry antibiotic-resistance genes)
What is gene conversion?
Phenomenon during recombination where alleles disappeared and were replaced by the allele on homologous chromosome. (i.e. AB, Ab --> AB, AB)
What are the start codons?
What are the stop codons?
UAG, UAA, UGA
What ends of the polypeptide correspond to the 5' to 3' ends of the mRNA strand?
Amini terminus and carboxyl terminus corresponds to 5'-3' of mRNA strand
How do you identify the location of the "cuts" in a transposon insertion?
Look for repeating elements along the same strand.
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