Unit 6 Flashcards
(101 cards)
1
Q
Generally speaking, what is the structure and function of DNA?
A
A long thin organic polymer in which the linear sequence of covalently linked nucleotide subunits encodes the genetic message
2
Q
Given the structures of the four common bases, identify each as a purine or pyrimidine (or describe their general structures)
A
Heterocyclic compounds, aromatic character:Purines - BIG. two nitro-carbon rings, one with 6C and the other 5C. Connected to pentose by nitrogen at “bottom” of 5C ring.Pyrimidines - SMALL. one n-c 6C ring, connected to the pentose by the nitrogen at the bottom of the ring.
3
Q
What are the characteristic components of a nucleotide?
A
1) Nitrogenous base (purine or pyrimidine)2) pentose3) one or more phosphate groups
4
Q
What are the components of a nucleoside?
A
A molecule consisting of a nitrogenous base and a pentose. NO PHOSPHATE.
5
Q
Convention: How are carbons numbered in the pentose ring of nucleotides?
A
Carbons are designated with ‘ or “# prime” carbon to distinguish them from the base carbons.
6
Q
Nucleotide structure: Which atom in the sugar is the base attached to?
A
1’ C
7
Q
Nucleotide structure: What bond connects the sugar and base?
A
N-beta-glycosyl bond.Formed from removal of H2O. Connects 1’ C to N-1 or N-9 in pyrimidines and purines, respectively.
8
Q
What is general deoxyribonucleotide structure?
A
Nucleotide (pentose, Pi, & N base). -H group at 2’ C of pentose. Still have 5’ and 3’ -OH available to bind phosphates and other groups.
9
Q
What is general ribonucleotide structure?
A
Nucleotide (pentose, Pi, & N base). Have -OH groups at 2’, 3’ and 5’ C.
10
Q
Draw structures representing:1) a 2’-deoxynucleoside2) a 2’-deoxynucleotide3) a 2’-deoxynucleoside 5’-di- or triphosphate
A
1) 2’-Deoxy = no -OH at 2’ C. NucleoSIDE = pentose and base.2) 2’-Deoxy = no -OH at 2’ C. NucleoTIDE = pentose, base, and one or more Pi groups, unspecified location.3) 2’-Deoxy = n -OH at 2’ C. NucleoSIDE + 5’-di or triphosphate = 2 or 3 phosphate groups connected at 5’ C.
11
Q
What characteristic defines DNA vs. RNA?
A
The pentose used in the nucleic bases:RNA has ribonucleotides.DNA has deoxyribonucleotides.
12
Q
What are the purine bases?
A
Guanine and Adenine (GA, big state that makes PURE orange juice).
13
Q
What are the pyrimidine bases?
A
Cytosine, Thymine, and Uracil. (CT is a small state with a lot of $$$).
14
Q
What functional group is at the 3’ position in ribo- and deoxyribonucleotides?
A
- OH
15
Q
Identify the atom that is found at the 2’ position in deoxyribonucleotides?
A
- H
16
Q
In DNA and RNA nucleotides, to which atom in the sugar is the phosphate bound?
A
5’ C
17
Q
Discuss the nucleotides found in DNA.
A
A, C, T, G. Never U.
18
Q
Draw a section of DNA (use the letters A, C, G, and T to represent the structure of the base). Point out:a. phosphodiester linkageb. sugar-phosphate backbonec. 5’ and 3’ endsd. overall charge
A
DO IT.
19
Q
What bond connects successive nucleotides?
A
Phosphodiester linkages. R-O-(PO2-)-O-R
20
Q
What is the overall charge of nucleic acid polymers?
A
Negative, because of the negative sugar phosphate backbone.
21
Q
What gives nucleic ACIDS (RNA, DNA) their acidic character?
A
The negatively charged backbone. The phosphate group acts as an acid much more so than the nitrogenous bases contribute basic character. They are protected on the interior of the duplex, usually.
22
Q
Convention: What direction are nucleic acid sequences written in?
A
ALWAYS 5’ to 3’.
23
Q
Distinguish between an oligonucleotide and a polynucleotide.
A
Oligonucleotide is short, or < 50 bases.Polynucleotides are longer than that.
24
Q
Discuss the hydrogen bonds that stabilize the DNA double helix.
A
Aromatic and relatively hydrophobic bases line the interior of the duplex. dBs give them slightly polar character and causes them to hydrogen bond to each other’s polar groups when complementary base pairs are aligned linearly as “wrungs” in the duplex ladder.A and T bond through 2 H bonds.G and C bond through 3.Bonding pairs are always purine/pyrimidine.
25
Describe how Hershey and Chase showed DNA to be the genetic material.
Expt. by Hershey and Chase:- Viruses were radioactively labelled so either proteins or DNA were detectable- Cells were infected and lysed.- Lysate was centrifuged and contents of pellet (the cells) were scanned for radioactive contentOutcome:- Only the virus with radioactive label in the DNA gave a radioactive cell pellet. ** The DNA was what the virus incorporated into the host and produced the genetically mediated viral infection. Radioactive caspids were in the supernatant (not what conferred the effect in cells).
26
How was DNA shown to be the genetic material?
The results of experiments by Avery, MacLeod, and McCarty: a non-infectious bacterium population was injected with the DNA of an infectious strain. The non virulent strain became infectious.
27
Why was the base composition (Chargaff's rules) important in suggesting a model for the structure of DNA?
Chargaff & co found that bases occurred in DNA in different proportions and that the proportions of certain bases were related. (%G = %C, %A = %T)This is supportive of the WC base pairing paradigm, where two certain bases occur jointly and therefore should compose equal proportions of the whole bp population.
28
What other information was crucial in the structure determination of DNA?
Franklin and Wilkins - x-ray crystallography and diffraction = helical with two periodicities (2 groove sizes)
29
Discuss the structure of B-form double-stranded DNA. (Point out the major and minor grooves, the sugar-phosphate backbones, and the stacked base pairs.)
Double helix formed by coiling complementary, anti parallel strands. Bases paired on interior (maintained by h-bonds and hydrophobic forces). 10.5 bp per turn = 36 Angstroms per turn and 3.4 Angstroms between bps.
30
Discuss the strand polarity in doublestranded DNA. What word is used to describe the polarity of the two strands?
Antiparallel.
| 5' to 3' is matched with a complementary, antiparallel strand going from 3' to 5'.
31
What base pairs are found in DNA? Discuss the key difference between these two base pairs.
Always purine paired with a pyrimidine.
C-G and A-T.
CG bases interact through 3 hydrogen bonds. AT interacts through 2.
32
What is the position of the sugar-phosphate backbone relative to the bases in B-form DNA?
Backbone forms the outer most layer of the helix and insulates the interacting base pairs in the core of the spiraling duplex.
33
Do Worked Example 8-1 on base pairing in DNA:
You are given the relative Adenine base composition of two gene sequences - 0.17 and 0.32 for bacteria Y and X. What are the relative other proportions of bases in these DNA samples? What assumptions have you made? Which species is likely thermophilic?
1) Y : 0.17 A = 0.17 T = 1 - (2*0.17) C = 1 - (2*0.17) G.
X: 0.32 A = 0.32 T = 1 - (2*0.32) C = 1 - (2*0.32) G.
2) Assumed that the DNA follows normal WC base pairing rules.
3) Y, bc a larger proportion of the bps are GC, which have more hydrogen bonds and stronger annealing (more resistant to high temperatures).
34
Discuss the types of bonds broken during denaturation.
Breakdown of duplex structure occurs because of hydrogen bond breakdown and base stacking interactions between base pairs. No covalent bond (phosphodiester) breakage involved.
35
What is the melting point (tm) of a given DNA segment? What is the relationship between tm and the base composition?
Tm = temperature at which 1/2 of the DNA in the sample has been denatured. Directly proportional to proportion of the DNA that is GC base pairs.
36
Define annealing.
The "re-zippping" of a denatured or partially denatured piece of DNA. When unwound segments of DNA spontaneous re-wind to give the intact duplex.
37
Define and distinguish between DNA replication, transcription, and translation.
Replication: DNA dependent synthesis of DNA (by DNAp)
Transcription: DNA dependent synthesis of mRNA (by RNAp)
Translation: RNA dependent synthesis of a polypeptide (by ribosome + tRNA)
38
What is a gene? How are genes named?
Gene is any segment of DNA that codes for some biosynthetic endproduct - INCLUDES INTRONS.
Bacteria gene naming convention: three letters, lowercase and italicized that reflect gene's apparent function.
39
What distinguishes a plasmid from a chromosome?
Plasmid is a small, extrachromosomal, self replicating element that is circularized. May be laterally passed and endow advantages or no change at all (common to prokaryotes).
Chromosome is a tertiary structure of DNA containing genetic information for the whole organism packed into nuclear, condensed arrangements in eukaryotes.
40
Is all eukaryotic DNA stored in the nucleus?
No: mitochondrial and chloroplast DNA is separate and replicates separately. Contributes to symbiotic theory for development of these energy metabolizing organelles.
41
What is an intron?
Intervening sequences in genes; noncoding regions of DNA that interrupt coding sequences, making for non linear or 1:1 transcription of DNA to mRNA. Essentially absent in prokaryotes.
42
What is an exon?
The coding region of a gene that is retained following post transcriptional processing. Make up about 1.5% of the human genome.
43
What is satellite DNA?
Another name for SSRs - simple sequence repeats. These are sequences about 10 bp long that are repeated many times in a section of a genome. Present in centromeres and telomeres. So called satellite DNA because when centrifuged, the DNA forms a band of a certain length distinct from the rest of the DNA, due to rigid base pair composition proportions.
44
What is a centromere?
A non transcribed segment of chromosomes that contains sequencing important for binding of proteins and mitotic spindles to enable proper sister chromatid assembly and separation upon division. Contain high proportion of A=T rich SSRs.
45
What is a telomere?
Noncoding ends of linear chromosomes that serve to stabilize the chromosomes by protecting the more interior coding sequences from incomplete replication that occurs with each round of replication.
46
What is supercoiling?
The coiling of a coil into a supercoil.
47
What is meant by "linking number"?
= Lk. A topological (does not change with deformation) characteristic of a fixed end (with proteins or circularized) piece of duplex DNA. If one strand is made rigid and straight, the linking number = the number of times the other strand makes a complete turn around the fixed strand. 1 "link" for each 360 degree rotation the strands around each other.
48
What is a "topoisomer"?
A DNA molecule with the same bp but a different linking number = overwound or underwound DNA
49
Discuss how over-winding or underwinding the DNA double helix leads to the formation of positive or negative supercoils, respectively.
Change in Lk by underwinding (decreasing number of turns per quantity of base pairs) or overwinding (increasing number of turns per quantity of base pairs) introduces strain into the relaxed DNA - requires strand breakage and ligation. The strain manifests in supercoiling in one direction or other to decrease or increase bps per turn, respectively = negative (left handed) or positive (right handed) supercoiling.
50
List two reasons why it is advantageous for cells to maintain their DNA in the underwound state.
1) Compaction by negative supercoiling.
2) Generation of potential in DNA that catalyzes duplex separation for transcription - strain introduced by strand separation during replication and transcription is negated by the underwound state.
51
What is a topoisomerase?
Enzymes that catalyze reactions to increase or decrease underwinding maintained in DNA by changing linking number of protein bound segments or circular DNA.
52
Differentiate between type I and type II topoisomerases in E. coli.
Type I
- Change Lk by 1 by breaking one strand and passing the other through.
- In E coli, Topoisomerase I and III are type one and both DECREASE underwinding (+ 1 Lk)
Type II
- Change Lk by + or - 2 by breaking both strands and passing another segment of two strands through.
- In E coli, topoisomerase II aka DNA gyrase USES ATP to introduce -2 Lk.
Balance btwn 2 opposing alterations maintains underwinding.
53
Describe how E. coli topoisomerase I removes negative supercoils, thereby increasing the linking number.
- Temporarily break phosphodiester bond between two residues by attacking alpha phosphate with Tyr residue at active site - forms temporary 5-phospohtyrosyl-protein linkage
- Passes other strand through breakage point.
- Ligates phosphodiester
54
Discuss the packaging of DNA in eukaryotes into nucleosomes.
DNA is packed in a "beads" on a string formation. Wrapped around nucleosomes, which are composed of 8 histone subunits. Subunits connected by small areas of unwound, linker DNA. Nucleosomes can vary in histone composition. Histone amino terminal tails extend outwards from complex. Each strand wraps about 1.67 times per nucleosome in a left handed, solenoidal, negative supercoil.
55
What is unique about the amino acid composition of histone proteins that is crucial to their DNA-binding function?
Histones have a high proportion of basic amino acids (Arg, Lys) that complement the negative backbone charge of DNA.
56
Discuss higher order organization of eukaryotic DNA.
In euchromatin, nucleosomes. In heterochromatin, methylated/condensed nucleosomes.
1) Nucleosome wound segments arranged into 30 nm fiber of further coiling in some regions, contain more H1
2) Arranged in loops attached to a proteinacious chromosome scaffold
3) Somehow arranged into thicker rosette like structures involving condensins and cohesins - Structural Maintenance of Chromosome (SMC) proteins
57
Explain what is meant by semi-conservative replication.
Each DNA strand is used as a template for the construction of a new complementary strand. Each daughter strand contains half of the original and is semi conserved.
- Proved by Meselson & Stahl - bacteria allowed to replicate once in presence of isotope N gave chromosomes of a single molecular weight = hybrid (not two types that would have arisen from non conservative replication).
58
Define what is meant by the term origin of replication.
Replication begins bidirectionally from a single initial sequence in the DNA. ORI signals the location for initiation of replication and is typically rich in A=T.
59
What is a replication fork?
The site where DNA is continually separated as the DNAp proceeds along the parent strand, simultaneously synthesizing two semi conserved daughter strands.
60
Discuss bi-directional replication.
Elongation always occurs 5' to 3', and proceeds in both directions along the parent strands from the ORI. When replication forks meet, original strands are separated into two daughters.
61
Discuss leading and lagging strand synthesis. Which strand is replicated continuously, and which discontinuously?
Since strand synthesis must occur from 3' to 5' along one strand at each replication fork (while elongation occurs 5' to 3'), those strand will be developed in discontinuous Okazaki fragments and are termed lagging strands. The opposite strands are read 3' to 5' - continually synthesized leading strands.
62
What is the direction of DNA synthesis by DNA polymerase?
Always elongated 5' to 3' (add to me)
63
In what direction is the template strand read in DNA synthesis by DNA polymerase?
Always read 3' to 5' (ready to drive and get the lead as the leading strand)
64
What are Okazaki fragments?
Fragments of new DNA generated in chunks from 5' to 3' along the lagging strand, for net synthesis of the new strand from 5' to 3'.
65
Distinguish between an exonuclease and an endonuclease.
Exonuclease - DNA nuclease (degrades DNA) that can only degrade starting from a strand terminal [Ex: 3' to 5' exo = proofreading - removes only from 3' or just synthesized end. 5' to 3' exo = erasure and redoing - removes only from 5' end]
Endonuclease - nuclease that degrades starting from internal sites. [Ex: restriction endonucleases]
66
What are the two "central requirements" of DNA polymerase for DNA polymerization?
1) Needs a 3' terminal to start adding on to (a primer terminus, from a primer)
2) Needs a template sequence to determine the nucleotide to add (original DNA template strand)
67
Discuss the reaction catalyzed by DNA polymerases.
Needs: deoxynucleoside-triphosphate, template, primer terminus (3' OH)
- Conserved Asp residues destabilize the negative charged alpha-beta phosphoanhydride bond
- 2 Mg2+ cofactors stabilize free O- on 3' C of previous sugar & the temporarily negative alpha-phosphate oxygens
- Nucleophilic attack of the alpha phosphate by the free 3' C-O of the previous nucleotide displaces the alpha-beta phosphoanhydride bond
- Creates pryophosphate byproduct
Thermodynamics:
- Phosophoanyhydride (and additional PPi phosphoanhydride) bonds broken
- Phosphodiester bond formed
- Stabilized by base stacking and hydrogen bonding in resulting duplex
68
What process drives the reaction toward the formation of products? Discuss in terms of Le Chatelier's principle.
Rapid breakdown of PPi to 2 x Pi by pyrophosphatases.
- Removing PPi product from polymerization rxn drives rxn towards products.
- Additional energy loss makes it essentially irreversible.
69
Define processivity.
Average number of nucleotides added by a bound and activated DNA polymerase before it dissociates. Characteristic of each polymerase & environment.
- Generally relates to how quickly the polymerization occurs for that polymerase.
70
Discuss how the geometry of the active site of DNA polymerase contributes to the fidelity of DNA replication.
Bp geometry - checks intial fit of incoming base into active site with given template base. Rejected before phosphodiester bond formation if failed.
71
Discuss proofreading by DNA polymerase.
3' to 5' exonuclease activity, either at insertion site or at post insertion, exonuclease site. Kinetic pause during translocation from insertion to post insertion site allows it to "check" the pairing. Can degrade the bond after it has been formed.
72
DNA pol I and DNA pol III:
1) 3' to 5' exonuclease activity (proofreading)
2) 5' to 3' exonuclease activity
3) Rates of polymerization
4) Processivity
#) characteristic - [DNA Pol I = small bacterial] vs [DNA pol III = big bacterial]
1) Proofreading - yes vs yes
2) Post completion editing (5' to 3') - yes vs. no
3) Rates - slow vs. fast
4) Processivity - low vs. high
73
Explain why DNA pol III is more processive than DNA pol I (E. Coli).
DNA Pol III has dual beta sliding clamps that allow it to polymerize more during one association. DNA Pol I binds to the DNA only by two "fingers" of the "hand."
74
What is the function of helicase in DNA pol?
Splits the DNA at the replication fork vertex. Separates strands so they can are accessible for the DNA pol.
75
What is the function of topoisomerase in DNA pol?
Relieve the over and underwinding stress placed before and after the replication fork, respectively.
76
What is the function of the DNA-binding proteins in DNA pol?
Bind to and stabilize the separated DNA strands, both to help helicase bind and to keep the strand separated during Okazaki fragment synthesis.
77
What is the function of primase in DNA pol?
Generates RNA primers for the initiation of elongation once for the leading strand and repeatedly for successive Okazaki fragments.
78
What is the function of DNA ligase in DNA pol?
Ligates the nicks in DNA following nick transition (RNA to DNA by DNA pol with 5' to 3' exonuclease activity) to seal breaks between fragments with phosphodiester bonds.
79
Discuss the sequence features at the origin of replication. (Note: Do not concern yourself with actual sequences, just the number and different types of repeated sequences present!)
At ORI:
1) Consensus DUE (DNA Unwinding element) Sequence - rich in A=T
2) 5 repeats of R sites - binding sites for DnaA DNA binding proteins
3) 3 additional DNaA binding I sites
4) Other activator protein binding sites (IHF, FIS)
80
Discuss the events that initiate DNA replication.
1) DnaA DNA binding proteins bind at R and I sites and associate in a way that strain is applied to DNA in form of right handed coil
2) Strain causes formation of replication bubble where DUE sequence splits
3) Other binding proteins associate and help DNA helicase bind to opening at DUE
4) Helicase binding separates replication forks bidirectionally and sets up replication bubble for binding by DNAp
5) Single stranded binding proteins (SSBs) stabilize & topoisomerases relieve strain
81
What is the function of the R and I sites? What happens at these sites that leads to unwinding of the double helix at the DUE?
AAA+ ATPases (ATPases associated with cellular activites) bind there and arrange themselves so strain causes denaturation at the DUE.
82
As the replication fork enlarges in DNA synthesis, what enzyme continues to be involved in unwinding the double helix?
Helicase = DnaB
83
What enzyme counteracts the positive supercoiling caused by the helicase, by introducing negative supercoils?
Topoisomerase II - a Type II topoisomerase that introduces negative coiling to relieve strain and allow easier procession of DNAp complex.
84
What protein binds to the resulting single-stranded DNA? Why is this important?
Single Strand Binding proteins (SSBs) - prevent annealing
85
What process is required before DNA synthesis can occur on the single-stranded template? Explain. What enzyme carries this out?
Primer needs to be made by Primase.
86
Why is RNA used as a primer instead of DNA for DNA polymerization?
RNA primers can be made by primase without a primer - it only needs a template.
87
What enzyme carries out the bulk of DNA replication in E. Coli after initiation, priming, and stabilization?
DNA Polymerase III - large, multisubunit polymerase with proofreading only, high speed, and high processivity. Moves behind fork generated continually by helicase.
88
Discuss how a dimer of DNA polymerase at the replication fork can copy both parental strands simultaneously. Discuss the role of the beta subunit.
Two core subunits, the clamp loader, and primase are complexed with helicase and proceed in one direction along the replication fork. Cores are held to DNA single strands by beta subunits (clamps)
1) DNA on lagging side forms a loop as synthesis.
2) Primase lays a new primer for each fragment and the clamp loader puts a new clamp at each new primer
3) when the lagging strand reaches the end of that primer, the whole beta clamp detaches, and the next prepped beta clamp is loaded
89
Name the enzyme that removes the RNA primer. Which activity is used for RNA primer removal?
DNA Polymerase I, using 5' to 3' exonuclease activity. Conducts nick transition.
90
Name the enzyme that then catalyzes the formation of phosphodiester bonds between the newly synthesized DNA fragments.
DNA ligase
91
Draw a diagram to explain how bidirectional synthesis works on image of a replication bubble.
DO ITTTT
92
What are telomeres?
Repetitive, non coding sequences at the ends of chromosomes that help stabilize the chromosomes by protecting interior coding sequences from being truncated after each chromosome replication.
93
Discuss why linear chromosomes cannot be entirely copied by lagging strand synthesis.
For the lagging strand side to be fully replicated, the last okazaki fragment must begin directly at the last basepair. This is very unlikely, so whatever is not included in the final "jump" will be cut of on one side of daughter chromosomes.
94
Name the enzyme that adds telomeric ends. What type of enzyme is it?
Telomerase, a specialized reverse transcriptase.
95
Discuss telomere addition. What serves as the primer for telomere addition? What serves as the template?
```
Primer = the 3' end of the DNA chromosome
Template = internal RNA sequence (appropriate AxCy sequence to make complementary TxGy extension from 3' end) that the enzyme uses as a RNA template to make the corresponding DNA addition to the end of the telomere.
```
96
In humans, what types of cells (germline or somatic) have telomerase?
Germline only.
97
Discuss the role of telomeres in cell senescence.
Linear reltionship between telomere length and cell longevity.
98
What is Base-excision Repair?
Process of removing a mutated site sequence by:
1) Location of damage site by appropriate endonuclease
2) DNA polymerase I reconstruction
3) DNA ligation
99
Discuss the spontaneous cytosine deamination reaction.
Cytosine deamination gives Uracil. The amine group is replaced by a carboxyl group in spontaneous mutation.
100
Discuss base-excision repair, naming each enzyme and discussing the function of each.
1) Uracil is detected as a mutation no matter what in DNA. Its base is cleaved off to give an abasic (apyridinic or AP) site/lesion by DNA glycosylase. 2) Base-excision repair removes the lesion with AP endonuclease.
3) DNA Polymerase I makes a new segment to replace the site with 5' to 3' endonuclease activity.
4) DNA ligase seals nicks.
101
List three key processes that contribute to the high fidelity of DNA replication.
1) Base geometry screening at active site, before phosphodiester bond formation.
2) DNAp III proofreading (3' to 5') activity, at active site and at post insertion, exonuclease activity site.
3) Later erasure and redoing by DNAp I endonuclease activity (fixes AP lesions and base mismatches)
