DNA Flashcards
DNA
- polymer made up of monomers called nucleotides
- forms a double helix that runs anti-parallel
- four bases: A, T, C, G
Nucleosides
- five carbon sugar (pentose) bonded to a nitrogenous base [base + sugar],
- formed by covalently linking the base to C1 of the sugar
- ex. ATP, ADP
Nucleotides
- contains 3 parts: 5-carbon sugar, nitrogenous base, phosphate group
- formed when one or more phosphate groups are attached to C5 of a nucleoside
- building blocks of DNA
- high energy compounds due to the repulsion between closely associated negative charges on phosphate groups
- ex. AMP, CMP, GMP
What are Adenine’s corresponding nucleosides? Nucleotides?
- Nucleosides: adenosine (deoxyadenosine)
- Nucleotides: AMP (dAMP), ADP (dADP), ATP (dATP)
What are Guanine’s corresponding nucleosides? Nucleotides?
- Nucleosides: guanosine (deoxyguanosine)
- Nucleotides: GMP (dGMP), GDP (dGDP), GTP (dGTP)
What are Cytosine’s corresponding nucleosides? Nucleotides?
- Nucleosides: cytosine (deoxycytosine)
- Nucleotides: CMP (dCMP), CDP (dCDP), CTP (dCTP)
What are Uracil’s corresponding nucleosides? Nucleotides?
- Nucleosides: uridine (deoxyuridine)
- Nucleotides: UMP (dUMP), UDP (dUDP), UTP (dUTP)
What are Thymine’s corresponding nucleosides? Nucleotides?
- Nucleosides: deoxythymidine
- Nucleotides: dTMP, dTDP, dTTP
Ribose
- found in RNA
- nucleic acid (sugar)
- presence of -OH group at C2 which makes it less stable than DNA
Deoxyribose
- found in DNA
- nucleic acid (sugar)
- presence of -H group at C2
What is the backbone of DNA composed of?
alternating sugar and phosphate groups
What is the overall charge of both RNA and DNA?
negative – b/c of the negative phosphate groups
What type of bonds form the sugar phosphate backbone of DNA?
3’-5’ phosphodiester bonds between nucleotides – phosphate group links the 3’ carbon of one sugar to the 5’ phosphate group of next sugar
DNA strands run _____ to one another
antiparallel
What direction do enzymes that replicate and transcribe DNA work in?
3’ to 5’
What is located at the 5’ end of DNA?
-OH or phosphate group boned to C5 of sugar
What is located at the 3’ end of DNA?
free -OH group on C3 of sugar
Purines
- aromatic heterocycle
- contain two rings in structure
- nucleic acid examples: adenine, guanine
Pyrimidines
- aromatic heterocycle
- contain one ring in structure
- nucleic acid examples: cytosine, thymine, uracil
Aromatic Compounds
Compounds must be:
- cyclic
- planar
- conjugated (alternating single and multiple bonds or lone pairs creating at least one unhybridized p-orbital for each atom in the ring)
- fairly unreactive due to delocalization of electrons that form pi electron clouds above and below ring
Huckel’s Rule
- rule followed by aromatic molecules
- has 4n+2 pi electrons
What are the key features of the Watson-Crick Model?
- two strands of DNA are antiparallel (one polarity 5’ to 3’ down and other 5’ to 3’ up)
- sugar-phosphate backbone is outside helix and nitrogenous bases on inside of helix
- A pairs with T via 2 H bonds, G pairs with C via 3 H bonds
- H bonds and hydrophobic interactions between bases provide stability to helix structure
Chargoff’s Rules
- purines and pyrimidines are equal in number in a DNA molecule
- amount of A=T and amount of C=G
B-DNA
- double helix of most DNA is a right handed helix so this is the form it is in
- turns every 3.4nm and contains 10 bases within that span
Z-DNA
- zigzag appearance
- left handed helix with a turn every 4.6nm and 12 bases within each turn
- form due to high GC content or high salt concentration
- highly unstable
Denaturation
- involves disruption of H bonding and base pairing
- none of the covalent links between nucleotides in DNA backbone break
- can be done by: heat, alkaline pH, chemicals like formaldehyde and urea
Reanneal
re-connection of single DNA strands by slowly removing the denaturing condition
DNA with larger percent of ___ and ___ bonds is more stable and takes more energy to break these bonds
C and G
Histones
- small basic proteins that DNA winds around to make chromosomes
- 5 histone proteins in eukaryotic cells
What are the 5 histone proteins?
- form the histone core: H2A, H2B, H3, H4 (two copies of each unit form the core)
- seals off DNA as it enters/exits the nucleosome: H1
Nucleoproteins
- proteins that associate with DNA
- most are acid-soluble and stimulate processes like transcription
Nucleosome
made up of 200 base pairs of DNA wrapped around histone core
Heterochromatin
- condensed during interphase
- transcriptionally silent
- compact nature makes it dark under light microscopy
Euchromatin
- uncondensed chromatin
- uncondensed during interphase
- genetically active – transcription occurs
- light under light microscopy
Telomeres
- repeated rounds of replication leads to shorter and shorter molecules of DNA with uneven ends
- so telomeres are long chain of noncoding DNA at ends of DNA that contain high GC-content to prevent unraveling [TTAGGG]
- uneven ends make them “sticky” so form “knots” which prevents linear DNA from being degraded at ends
- lengthens lifespan of eukaryotic cell by protecting fidelity of linear DNA
- when telomeres get too short the cell undergoes apoptosis
Telomerase
- replaces TTAGGG sequence at end of DNA
- doesn’t require Okazaki fragments
Centromeres
- regions of DNA that separate the arms of chromosomes
- composed of heterochromatin
- join sister chromatids until anaphase in cell division (S phase until anaphase)
- contain high GC-content to maintain strong bond between chromatids
Replisome
- replication complex
- set of specialized proteins that assist the DNA polymerases
List the steps of involved in DNA Replication
- DNA unwinds at origins of replication – replication forks form on both sides of the origin, increasing replication efficiency
- Topoisomerase moves ahead of the replication fork and keeps single strands stable
- Helicase creates the replication fork but also unwinds and separates parent strands after creation of fork
- Single-Strand Binding Proteins preserve helicases’ work
- Primase adds RNA primers to parental strand where polymerase can bind
- DNA Polymerase III moves along parent strand in 3’ to 5’ direction while synthesizing a new DNA strand in the 5’ to 3’ direction
- DNA Polymerase I removes the RNA Primers and adds the correct nucleotides (lagging strand)
- DNA Ligase joins the okazaki fragments together on the lagging strand (lagging strand)
Describe Eukaryotic cell origins of replication
- each eukaryotic chromosome has one linear molecule of double-stranded DNA with multiple replication origins
- chromatids remain connected at centromere as replication forks move toward each other and sister chromatids are created
- slower process than prokaryotes
Describe Prokaryotic cell origins of replication
- bacterial chromosome is a closed double-stranded circular DNA molecule with a single origin of replication
- 2 replication forks move away from each other in opposite directions around circle
- results in production of two identical circular molecules of DNA
Helicase
- responsible for unwinding DNA
- generates 2 single stranded template strands ahead of the polymerase
- enzyme that breaks H bonds between nitrogenous bases
Single-Stranded DNA Binding Proteins
- binds the single stranded DNA and prevents it from re-annealing with the parent strand
- proteins that hold the unwound DNA strands apart by hydrogen bonding to the free purines and pyrimidines which prevents reassociation of DNA strands and degradation of DNA by nucleases
DNA Topoisomerases
- introduce negative supercoils when working ahead of helicase by knicking one or both strands which alleviates supercoiling
- relaxes torsional pressure then reseals cut strands
- breaks phosphodiester bonds in DNA backbone
Leading Strand
- copied in a continuous fashion in the same direction as the advancing replication fork
- reads parental strand in 3’ to 5’ direction
- RNA Primer is added to 3’ end via DNA Primase which tells DNA Polymerase where to bind
- DNA Polymerase III adds nucleotides at 3’ end, synthesize daughter strands of DNA in 5’ to 3’ direction
Lagging Strand
- copied in direction opposite to the direction of the replication fork
- DNA Primase starts near replication fork and moves away, and continuously adds small RNA Primers (~10 nucleotides) away from the replication fork
- DNA Polymerase III begins synthesizing daughter strands of DNA in 5’ to 3’ manner (reads parental strand from 3’ to 5’ end) which leads to the creation of okazaki fragments
- DNA Polymerase I removes RNA primers and adds the corresponding nucleotides
- DNA Ligase joins the Okazaki Fragments together
What are the different DNA Polymerases used in Eukaryotes?
- RNase H
- alpha (α)
- delta (δ)
- gamma (γ)
- beta (β)
- epsilon (ε)
What is the function of both DNA Polymerase α and δ?
add nucleotides – extend DNA
What is the function of DNA Polymerase γ?
replicates mitochondrial DNA
What are the functions of both DNA Polymerase β and ε?
function in processes of DNA repair
What are the functions of both DNA Polymerase δ and ε?
assisted by the PCNA protein which assembles into a trimer for form the sliding clamp which helps strengthen the interaction between DNA Polymerase and template strand
What are the key features of DNA Polymerases?
- need a template strand
- can only add nucleotides at 3’ end
- require a short stretch of nucleotides (primer) to start
- intrinsic proofreading function -> remove vast majority of “wrong nucleotides”
What are the 3 requirements for DNA replication?
- dNTPs (elongate new strand)
- ATP (necessary for polymerase function)
- primer
What are the different DNA Polymerases used in Prokaryotes?
- I
- III
DNA Polymerase I
- removes RNA primers and adds the correct nucleotides
- has exonuclease activity
DNA Polymerase III
adds nucleotides – does the main synthesis
What enzyme corresponds to each step in Eukaryotic DNA replication:
- Unwinds DNA double helix
- Stabilizes unwound DNA strands
- Synthesizes RNA Primers
- Synthesizes DNA
- Removes RNA Primers
- Replaces RNA with DNA
- Joins Okazaki fragments
- Removes positive supercoils
- Synthesis of Telomeres
- Helicase
- Single-Stranded DNA Binding Protein
- Primase
- DNA Polymerase α, δ, ε
- RNase H (DNA Polymerase β)
- DNA Polymerase δ
- DNA Ligase
- DNA Topoisomerase
- Telomerase
What enzyme corresponds to each step in Prokaryotic DNA replication:
- Unwinds DNA double helix
- Stabilizes unwound DNA strands
- Synthesizes RNA Primers
- Synthesizes DNA
- Removes RNA Primers
- Replaces RNA with DNA
- Joins Okazaki fragments
- Removes positive supercoils
- Synthesis of Telomeres
- Helicase
- Single-Stranded DNA Binding Protein
- Primase
- DNA Polymerase III
- DNA Polymerase I
- DNA Polymerase I
- DNA Ligase
- DNA Topoisomerases (DNA gyrase)
- N/A
Cancer Cells
- proliferate excessively because divide without stimulation from other cells
- not subject to normal cell proliferation controls
- have a way to extend their telomeres
Metastasis
migration of cancer cells to distant tissues by blood or lymphatic system
Tummor Suppressor Genes
- p53 and Rb
- encode for proteins that inhibit the cell cycle or participate in DNA repair processes
- mutations of these genes result in loss of tumor suppression and cancer
What intrinsic function of DNA Polymerase occurs during replication?
proofreading
-excises incorrectly matched bases (unstable H bonds)
and replaces with correct nucleotide
Mistmatch Repair
- catches whatever DNA Polymerase misses
- also removes and replaces mismatched nucleotides
- occurs during G2 phase of cell cycle using enzymes encoded for by genes MSH2 and MLH1
Nucleotide Excision Repair
- occurs during G1 and G2 of cell cycle
- repair occurs when UV light induces formation of dimers between adjacent thymine residues in DNA which distorts shape of double helix and interferes with DNA replication
What are the 4 steps involved in Nucleotide Excision Repair?
- specific proteins scan and recognize lesion because of bulge in DNA strand
- Excision Endonuclease nicks phosphodiester backbone of damaged strand on both sides of dimer and removes the defective oligonucleotide
- DNA Polymerase fills in gap by synthesizing DNA using undamaged strand as template
- nick in strand is sealed by DNA Ligase
Base Excision Repair
- fixes nondeforming lesions of the DNA helix (such as cytosine deamination) by removing the base
- occurs during G1 and G2 phase of cell cycle
- corrects lesions that are small enough not to distort helix
What are the 3 steps involved in Base Excision Repair?
- affected base recognized and removed by Glycosylase enzyme, leaving behind an apurinuc/apyrimidine (AP Site) site
- AP Site recognized by AP Endonuclease that removes the damaged sequence from the DNA
- DNA Polymerase and DNA Ligase compete process
Recombinant DNA Technology
- allows a DNA fragment from any source to be multiplied by gene cloning or PCR
- it is DNA composed of nucleotides from two different sources
DNA Cloning
- technique that can produce large amounts of a desired DNA sequence
- introduces a fragment of DNA into a vector plasmid (piece of nucleic acid) forming a recombinant vector
Restriction Enzymes
enzyme produced by bacteria that recognizes and cuts nucleic acids at very specific sequences
Complete Complementary
occurs when each nucleotide correctly binds with its pair
Partial Complementary
occurs when some nucleotides are matched with the incorrect pair and some are matched correctly
No Complementary
no nucleotides are correctly paired
What is a nuclease?
enzyme that cuts nucleic acids
How do endonucleases differ from exonucleases?
Endonucleases cut in the middle of a DNA chain, while exonucleases nibble away the nucleotides at the ends of the DNA chain
What are “sticky” ends of DNA?
unpaired, single-strand extensions of DNA that are ready to bind with complementary codons (= sequence of 3 adjacent nucleotides)
DNA Libraries
large collections of known DNA sequences produced via cloning
cDNA (complementary DNA) Libraries
- constructed by reverse-transcribing processed mRNA
- lacks noncoding regions (introns)
- only includes genes expressed in the tissue from which the mRNA was isolated
Answer the following questions about cDNA libraries:
- Source of DNA
- Enzymes to make library
- Cloned genes are complete sequences
- Cloned genes have introns
- Promoter and enhancer sequences present
- Gene can be expressed in recombinant proteins
- Used for gene therapy or making transgenic animals
- mRNA
- reverse transcriptase, DNA Ligase
- Yes
- No
- No
- Yes
- Yes
Answer the following questions about Genomic libraries:
- Source of DNA
- Enzymes to make library
- Cloned genes are complete sequences
- Cloned genes have introns
- Promoter and enhancer sequences present
- Gene can be expressed in recombinant proteins
- Used for gene therapy or making transgenic animals
- chromosomal DNA
- restriction endonuclease, DNA ligase
- Not necessarily
- Yes
- Yes
- No
- No
What are Nucleic Acids?
linear polymers built from monomeric subunits, put nucleotides together into DNA and RNA
What is more stable, DNA or RNA?
DNA
this is due to the OH group on the C2 group in RNA that is very reactive
What bases are considered Purines?
- PURE AS GOLD*
- Adenine
- Guanine
What bases are considered Pyrimidines?
- pyramids CUT*
- Cytosine
- Uracil
- Thymine
What makes Nitrogen lone pairs found in nucleic acids basic vs. aromatic?
- Basic: lone pairs that are found on N’s that are in a ring, with a double bond
- Aromatic: lone pairs that are found on N’s that are in a ring, with only single bonds
___ on pyrimidine attaches to ___ of sugar via a __-____ link.
- N1
- C1’
- beta-glycosidic
___ on purine attaches to ___ of sugar via a __-____ link.
- N9
- C1’
- beta-glycosidic
T pairs with __
A
G pairs with ___
C
___ bonds occur between nucleic acid pairs
Hydrogen
What is a hydrogen bond donor?
a hydrogen attached to N, O, or F
What is a hydrogen bond acceptor?
a lone pair of elections
How many hydrogen bonds occur between adenine and thymine?
2
How many hydrogen bonds occur between guanine and cytosine?
3
Which base pair is more stable, A & T or G & C?
G & C because they have more hydrogen bonds
The sugar-phosphate backbone of DNA is ____ charged and ____
-negatively
-polar
[negative charge comes from phosphate and polarity comes from sugar]
The nitrogenous bases that make up the inside of DNA are largely __-____
-non-polar
Watson and Crick Model of DNA
(1) 2 helical polymer strands that are coiled in a right hand sense
(2) strands are anti-parallel to one another
(3) sugar and phosphate on outside are polar and charged, while the bases inside are non-polar
(4) bases are nearly perpendicular to axis which allows for pi stacking
(5) bases are separated by 3.4 Å
(6) helix repeats every 34 Å (10 nucleotides per turn)
(7) bases associate in pairs (A&T, G&C, A&U for RNA)
Nucleic acid bases are separated by ___ Å.
3.4
DNA helix repeats every __ Å which correlates to ___ nucleotides per turn
- 34
- 10
What 5-Carbon sugar is found in DNA?
deoxyribose
What 5-Carbon sugar is found in RNA?
Ribose
Overall DNA is ____ charged.
negatively
DNA backbone is held together via ______ bonds that form between the ____ and the ____ groups.
- phosphodiester
- sugar
- phosphate
`DNA double helix has a diameter of ___ Angstroms.
20
The 5C sugar found in DNA is deoxy at the C’-__ position.
C’-2
deoxy sugar is a sugar that has had a hydroxyl group replaced with a H atom
RNA is ____ charged.
negatively
Primase
- synthesizes RNA primers
- puts RNA primers down on DNA where polymerase can bind
- primers give the DNA something to stick to (not needed in RNA because it can just start)
