Exam 2 Flashcards
Complexity and DNA content of organisms
There is no link between the amount of DNA content and complexity of organisms
Pyrimidines
Cytosine, Uracil, and Thymine
Purines
Adenine and guanine
Nucleoside
Nitrogenous base + a pentose sugar
β- Glycosidic bond (in nucleosides)
C1 of sugar is linked to N9 of a purine or N1 of a pyrimidine
What sugar is found in DNA
2-deoxy-D-ribose
Furanose
5-membered carbohydrate ring with 4 carbons and oxygen. Formed by the reaction of the C4 hydroxyl group with the terminal aldehyde.
Pyranose
6-membered carbohydrate ring with 5 carbons and 1 oxygen. Formed by the reaction of the C5 hydroxyl group and a terminal aldehyde
Which is more stable? DNA or RNA
DNA because fewer secondary structures are available and the 2-OH is susceptible to hydrolysis
Which conformation is favored in both pyrimidine and purine nucleosides?
Anti conformation
Nucleotides
When a phosphoric acid is esterified to the C5 OH of a nuceloside
Deoxyadenylate
Deoxyadenosine-5-monophosphate, A, dA, dAMP
Deoxyguanylate
Deoxyguanosine-5-monophosphate, G, dG, dGMP
Deoxythymidylate
Deoxythymidine-5-monophosphate, T, dT, dTMP
Deoxycytidylate
Deoxycytidine-5-monophosphate, C, dC, dCMP
Adenylate
Adenosine-5-monophosphate, A, AMP
Guanylate
Guanosine-5-monophosphate, G, GMP
Uridylate
Uridine-5-monophosphate, U, UMP
ytidylate
Cytidine-5-monophosphate, C, CMP
Post-DNA synthesis modification in eukaryotes
5-methylation, activates genes
Post-DNA synthesis modification in bacteria
N6-methylation, prevents immune degradation
Inosine
Deaminated adenosine. Minor nucleoside found sometimes in the wobble position of anticodon tRNA.
Pseudouridine
Found in eukaryotes and eubacteria. Made from the enzymatic isomerization of uridine after transcription. Useful to stabilize the structure of tRNA and folding of rRNA
Functions of different nuceloside-5-triphosphates
ATP is used for energy metabolism, GTP drives protein synthesis, CTP drives lipid synthesis, and UTP drives carbohydrate metabolism
Phosphodiester bonds
Link the 3’ oxygen and phosphate to the 5’ carbon
Directionality of nucleic acids
Bases are added and the strand is read in the 5’ to 3’ direction
Differences between DNA and RNA
DNA is more stable, only has one purpose, has 2-deoxyribose sugar, and contains thymine. RNA has 4 different types and purposes, ribose sugar, and urasil.
Why does DNA contain thymine?
Cytosine can spontaneously deaminate to form uracil, so thymine is just uracil with a 5-methyl group to prevent repair enzymes from recognizing urasil as mutated cytosine
Rosalind Franklin
Used X-ray fiber diffraction to identify the double helical structure of DNA called crosses and diamonds
Differences between the 3 forms of DNA
A-DNA has a shorter and broader helical turn than B-DNA and Z-DNA has a repetitive alternating pyrimidine-purine sequence which causes left-handedness (inverted B, basically).
Chain termination method of Sanger sequencing
Dideoxynucleotides are fluorescently labeled and then used by a DNA polymerase to create a DNA strand between two primers. Each ddNTP corresponds to a specific color and size.
Chemical cleavage method of DNA sequencing
ssDNA is labeled with P-32 on one end and then chemically cleaved between a specific base, creating differently sized DNA fragments. DNA can then be purified and read directly on a gel
Tertiary DNA structure
Duplex DNA can only have 10 bp per turn. Circular DNA can either be supercoiled, underwound, or overwound.
L=T+W
Linking number equals the number of helical turns (twist) plus the number of times the double helix crosses over itself (writhe).
DNA gyrase
A topoisomerase which creates a negative supercoil to separate circular DNA strands and localized unwinding. Inhibition of gyrase is a common antibiotic (ex: cipro)
Nucleosomes
2 turns of DNA supercoiled in a solenoid fashion around a histone core octamer (6 nucleosomes per turn)
DNA replication is…
Semiconservative, bidirectional, and semidiscontinuous
Which strand is the leading strand? Lagging strand?
the 5’ to 3’ strand is leading while the 3’ to 5’ strand is lagging
DNA replication in bacteria
dnaA is the major initiator protein, dnaB is helicase, dnaG is primase, and dnaC is helicase inhibitor.
DNA pol 1 - E. coli
First DNA polymerase ever discovered which catalyzes the extension of Okasaki fragments. Has 3’ and 5’exonuclease activity: 3’ acts as proofreading and 5’ removes primers
DNA pol 3 - E. coli
The real DNA polymerase which catalyzes DNA replication from RNA primers. Has 10 different subunits, alpha is the polymerase, epsilon is 3’ exonuclease, and theta is the holoenzyme assembly
DNA ligase - E. coli
Seals the nicks between Okazaki fragments
How does DNA replication occur on eukaryotic chromosomes
Different regions are replicated at different times during S phase. Highly condensed chromatin replicates late while less condensed chromatin replicates early. Demonstrated using BrdU
Autonomously replicating sequence
Sequences on eukaryotic chromosomes which are able to act as the origins of DNA replication.
Origin recognition complex
Comprised of the Pre-RC and post-RC complexes present at the replicators throughout the cell cycle
Which proteins are in the pre-RC?
Cdc6, Cdt1, and MCM and more depending on the cyclin-CDK levels
S. cerevisiae
Known replicator which binds to 11 bp ACS sequences
S. pombe
Known replicator which binds to 9 repeated AT motifs at the N-terminus based on recognition of A-rich DNA
D. melanogaster
Known replicator which undergoes ATP-dependent binding to the chorion locus with all 6 of its subunits.
3 Potential mechanisms of ORC binding control
1) stabilization by other replication factors 2) recruitment of replicators by ORC recruiting factor sequences 3) nucleosome positioning factors which alter chromatin and DNA accessibility on ORCs
What are the 7 steps for the sequential MCM2-7 loading model by ORC and cdc6 ATPases
1) ORC and Cdc6 bind to DNA 2) this opens the Mcm2-7 receptor to ctd1 3) the first helicase loading 4) co loaders release 5) the second helicase loading 6) co loader and initiator release 7)DNA melting and helicase activation
Yeast CMG complex
GINS, Cdc45, and MCM create a complex at DNA replication forks. The CMG complex plus other proteins make up the RPC
Initiation of DNA replication in eukaryotes
The pre-RC binds to the origin and is followed by MCM2-7 loading during G1 phase. During S phase, Sld2 and Sld3 proteins are phosphorylated to recruit DNA polymerase. MCM complexes serve as helicases
Helicase in bacteria vs humans
Bacteria- dnaB Humans- MCM proteins
Primase in bacteria vs humans
Bacteria- dnaG Humans- primase subunit of pol alpha
Main polymerase in bacteria vs. humans
Bacteria- alpha unit of pol III; Humans- pol delta (δ)
Proofreading exonuclease in bacteria vs. humans
Bacteria- epsilon unit of pol III; Humans - parts of pol delta (δ) and pol epsilon
Clamp loader of bacteria vs. humans
Bacteria- gamma subunit; Humans - RF-C
SS DNA binding protein
Bacteria- SSB; Humans- RP-A
Difference between protein components of eukaryotic and prokaryotic replication machines
They have the same basic functions, but eukaryotic ones have more subunits
Gap phases
Between S and M phases of the cell cycle which allow cells more time to grow and ensure conditions are suitable for the next stage
Metaphase checkpoint Qs
Are all the chromosomes attached to the spindle?
3 Major cyclins
G/S-cyclin-Cdks trigger progression through the start checkpoint (assess environment & DNA damage). S-cyclin-CDKs trigger DNA replication machinery (assess DNA damage). and M-cyclin-CDKs trigger mitosis machinery (asses DNA damage and unreplicated DNA)
Mechanism of Cdk activation
During the inactive state, the T-loop blocks the active site. Once the cyclin binds, it becomes partially active with the t-loop still present but in a different conformation. The phosphorylation of Cdk by CAK moves the T-loop and allows substrate binding to proceed with the cell cycle.
Measures to ensure there is only one DNA replication initiation per cell division
1) licensing of replication origins and 2) the activation of replications at the origins during S phase
prevention of re-replication study conclusions
S phase cytoplasm has a factor which drives G1 nuclei straight into synthesis but this factor is removed in G2. G2 nuclei are in refractory, so adding this factor does not cause a secondary S phase
Actual mechanisms of preventing re-replication
Inhibition of the pre-RC by 1) an inhibitory geminin-Cdt1 complex 2) Ctd1 &c Cdc6 degradation in S phase by phosphorylation 3) High activity of Cdk in G2 and M inactivates the pre-RC components
Geminin
Protein which inhibits DNA replication through Cdt1 and prevents MCM protein movement into pre-RC primarily during S, G2 and M phases but is degraded during metaphase/anaphase.
Order of chromosome packing (increasingly larger)
Nucleosomes, filament, DNA loops, multiband unit, chromatid
PCNA
Bridges genetic and epigenetic inheritance as a DNA polymerase processivity factor and a chromatin methylation/deacetylation platform
Endoreplication
DNA replication during S phase without the completion of cytokinesis
Polyteny
repeated DNA replication without the formation of new nuclei
Polyploidy
Mitosis without cytokinesis
Vertebrate telomere consensus sequence
TTAGGG
Telomerase
RNA dependent DNA polymerase which maintains telomere length by restoring telomeres at the 3’ end
Reverse transcriptase enzyme activities
RNA-directed DNA polymerase forms cDNA, RNAse H degrades the RNA:DNA hybrids, and DNA-directed DNA polymerase makes a DNA duplex
Reverse transcriptase primers
tRNA molecule captured from the host
First approved drug for AIDS
AZT which binds to HIV reverse transcriptase and replaces dTTP with AZTTP, which blocks further chain elongation
Non-homologous recombination
Low frequency occurrence of very different nucleotide sequences recombining
Transposition
Enzymatic insertion of a mobile segment of DNA (transposon)
Holliday model for homologous recombination
1) DNA duplexes align, 2) single-stranded nicks/cuts at homologous sites on two chromosomes 3) strand invasion or crossover through partial unwinding and base pairing with the other duplex 4) cross-stranded intermediate (Holliday junction) occurs due to ligation 5) branches migrate by unwinding and rewinding of the linked duplexes 6) Resolution of the Holliday junction by additional nicks
EW cleavage of Holliday junctions
Negative strands are cleaved, causing a patch recombinant heteroduplex
NS cleavage of Holliday junctions
Positive strands are cleaved, causing a splice recombinant heteroduplex
RecBCD
A helicase/nuclease which initiates recombination in E. coli. Binds to the recombinational hotspot Chi site of DNA and causes rabbit ear of ssDNA loops due to slow release.
RecA
Binds to the 3’ end to forms nucleoprotein filaments for strand invasion and homologous pairing during recombination. This filament causes the homologous pairing of dsDNA and strand invasion. RecA also binds to the other strand of DNA to find the homologous end.
RuvA, RuvB, & RuvC
Drive branch migration and help to resolve a Holliday junction into recombination products. RuvA catalyzes junction binding and RuvB catalyzes branch migration, and RuvC is an endonuclease which cuts the DNA strands
Class 1 Retrotransposons
Reverse transcriptase creates DNA from RNA and inserts it into the genome at a new position (copy & paste)
Class 2 DNA transposons
Transposase enzyme creates a cut in the host DNA which is then filled in and the inverted repeat transposon inserts itself.
Ames test
Uses salmonella that is unable to synthesize Histidine, you add a compound to see if it will restore the ability to synthesize His.
Direct reversal repair
Repair system that returns DNA to its proper chemical state
Photolyase
Enzyme used to repair pyrimidine dimers
DNA glycosylase
Base excision repair removes a damaged base, creating an AP site
AP endonuclease
Removes several residues that are chemically damaged and the gap is repaired by DNA polymerase and ligase
Nucleotide excision repair
Repairs larger regions of damaged DNA than base-excision
ABC excinuclease in bacteria
Contains UvrA, UvrB, and UvrC to hydrolyze lesions on 5th bond of 3’ end and 8th bond of 5’ end to remove 12-13 nucleotides.
Eukaryote nucleotide excision repair
Hydrolysis of the 6th bond on the 3’ end and the 22nd bond on the 5’ end to remove 27-29 nucleotides
Mismatch repair
Excises mispaired UNmethylated region and replaces it with methylated post-replication strand
Transition point mutation
Purine is replaced with a purine or pyrimidine replaced for another pyrimidine
Transversions
A pyrimidine is replaced with a purine or vice versa
Base analogs
Mutations can be induced by altered bases such as 5-bromouracil or 3-aminopurine
