BIOL 280 - Exam 4

What is a phosphodiester bond and how does it form the DNA backbone?

A phosphodiester bond is a covalent linkage between the 3′ OH group of one nucleotide and the 5′ phosphate group of the next nucleotide.
This creates the sugar-phosphate backbone of DNA.

• Repeats along the strand → forms a continuous chain

• Leaves nitrogenous bases free for hydrogen bonding

What is DNA polarity and how is it represented?

DNA has directionality (polarity):

• One end = 5′ end (free phosphate group)

• Other end = 3′ end (free OH group)

Sequences are always written 5′ → 3′
Example: pACGTA

• “p” = phosphate at 5′ end

• “OH” (if shown) = 3′ end

This direction is critical for DNA replication and synthesis

nucleotides are linked together via?

phosphodiester bonds

What are hydrogen bonds in biological systems and what factors affect their strength?

Hydrogen bonds occur between a hydrogen donor (X–H, where X = O or N) and a hydrogen acceptor (O or N with lone pairs).

Strength depends on geometry:

• Linear (straight) hydrogen bonds → stronger

• Angled hydrogen bonds → weaker (common in proteins due to structural constraints)

Overall: Hydrogen bonds are weak individually but collectively stabilize biological structures.

hydrogen bonding helps with what kind of interactions in DNA?

base pairing

base pairs are purine and pyrimidine what are the two base pairings for DNA? and how many hydrogen bonds are associated with the pairings

• G pairs with C —> 3 hydrogen bonds

• A pairs with T —> 2 hydrogen bonds

BLANK allows for information to be copied

complementarity

in terms of the structure of DNA the sugar phosphate backbond is on the BLANK and the bases are stacked on the BLANK

• sugar phosphate backbone is stacked on the outside

• bases are stacked on the inside

DNA —> DNA is

replication

what experiments were utilized to get the three dimensional structure of DNA?

in 1953, Watson and Crick postulate a model of DNA structure based on X-ray diffraction experiments of Rosalind Franklin and Maurice Wilkins. This model could account for a vast amount of experimental observations that had been accumulated.

DNA forms a right or left handed helix?

What are the types of grooves and please describe them

• DNA forms a right handed doubel helix

• Major groove: wide and shallow —> important for protein binding

• minor groove: narrow and deep

The DNA double helix is stabilized by

• hydrogen bonding — between base pair

• stacking interactions

what are the differences between the B form and A form of DNA?

B form DNA:

• most stable

• found in solution

• 10.5 bp per turn

• right handed

A form DNA:

• DNA-RNA, RNA-RNA helix

• found in solution

• 11 bp per turn

• right handed

A form DNA is more compacted

what is a Z form helix

left handed

what leads to the denaturation of DNA?

high temperature or pH

What is DNA denaturation and how is it measured?

• Denaturation: DNA goes from double helix → random coil (single strands)

• Measured by UV absorbance

• Denaturation → ↑ UV absorbance

• Due to loss of base stacking interactions

• Called the hyperchromic effect

• double helix has stacked bases so it would have LESS UV absorbance while single strand DNA will have more UV absorbance since it has unstacked bases and more exposure to light so it can absorb more

What is melting temperature (Tm) in regards to denaturation? it tells us what about DNA

• Tm: temperature where 50% of DNA is denatured

• Half helical, half random coil

• Used to compare DNA stability

How does GC content affect DNA stability?

• More G≡C → higher Tm → more stable DNA

• Less G≡C → lower Tm → less stable DNA

• On graph: curve with higher Tm = higher %GC

so higher amounts of GC require higher amounts of temperature to denature since the GC bp has 3 H bonds compared to 2. hence higher Tm compared to another can tell us that we have higher GC content than the other.

higher GC bp amount requires BLANK temperature to denature

higher

what is the overall structure of RNA?

• single stranded molecule

• forms a single stranded helix (not double helix like DNA)

• can fold back on itself via base pairing

how does RNA form secondary structure?

• intramolecular base pairing (within same strand)

• creates double stranded regions

• these regions adopt an A-form helix

what secondary structure element is this?

hairpin

what secondary structure element is this?

internal loop

what secondary structure is this?

bulge: one or more unpaired bases on one side only

• causes a distortion in the helix

what secondary structure is this?

single stranded regions in RNA: regions with no base pairing

what type of helix do RNA double stranded regions form?

A form helix

• more compact and rigid than B form DNA

• occurs in RNA hairpins and stems

what base pairs occur in RNA?

• canonical pairs: A-U, G-C

• Non canonical pair : G-U (wobble)

• G-U pairing helps RNA form complex secondary structures

what is RNase P?

an RNA enzyme (ribozyme) that cleaves RNA (tRNA processing)

how do DNA binding proteins recognize specific sequences?

• Primarily through major groove interactions

• Hydrogen bonding with exposed base edges

• Major groove provides more chemical information than minor groove
  → Enables sequence-specific recognition without unwinding DNA

why is sequence specific DNA recognition important?

• allows proteins to identify specific DNA sequences

• essential for information transfer processes:

  • transcription

  • replication

  • DNA repaire

• ensures correct genes are regulated or accessed

How does the major groove enable sequence-specific recognition?

• Distinct donor/acceptor patterns for each base pair

• Can distinguish:

  • AT vs TA

  • GC vs CG

• Provides maximum chemical information
  → Main site for DNA-binding protein recognition

What information can be obtained from the minor groove?

• Limited chemical information

• Can distinguish:

  • AT/TA vs GC/CG

• Cannot distinguish AT from TA
  → Less useful for precise sequence recognition

Why is the major groove more important than the minor groove?

• Major groove exposes unique H-bonding patterns for each base pair

• Minor groove patterns are more similar/redundant
  → Proteins bind major groove for accurate sequence reading

How do proteins recognize DNA sequences?

• Amino acid side chains form hydrogen bonds with DNA bases

• Interact with donor/acceptor groups in grooves (mainly major groove)

• Recognition depends on matching H-bond patterns
  → Enables sequence-specific DNA binding

Which amino acids commonly interact with DNA bases? what is the mneomnic we can use?

• Asn (asparagine)

• Gln (glutamine)

• Glu (glutamate)

• Lys (lysine)

• Arg (arginine)

• These side chains can act as H-bond donors and/or acceptors

“Naughty Queens Grab Large Apples”

• N → Asn (Asparagine)

• Q → Gln (Glutamine)

• G → Glu (Glutamate)

• L → Lys (Lysine)

• A → Arg (Arginine)

What determines sequence specificity in DNA-protein interactions?

• Each base pair has a unique H-bonding pattern

• Proteins use a specific arrangement of amino acids

• Must match the pattern of bases in sequence order
  → “Reading” DNA = matching amino acid side chains to base patterns

DNA binding proteins typically bind where?

• Generally bind DNA in the major groove — where it can recognize the sequence the beest

  • alpha helix fits nicely into the wide major groove

• certain DNA binding motifs are common

  • helix turn helix

  • zinc finger

  • homeodomina

  • leucine zipper

  • basic helix loop helix (BHLH)

What is the helix–turn–helix DNA-binding motif and how does it recognize DNA?

• Protein motif with two α-helices connected by a turn

• One helix = recognition helix

• Recognition helix fits into the major groove of DNA

• Amino acid side chains interact with base pairs via H-bonding
  → Allows protein to read specific DNA sequences without unwinding DNA

What is the zinc finger DNA-binding motif and how does it recognize DNA?

• Protein motif stabilized by a coordinated Zn²⁺ ion

• Zn²⁺ held by 4 Cys OR 2 Cys + 2 His residues

• Contains a recognition helix that inserts into the major groove

• Each “finger” recognizes a few base pairs

• Multiple zinc fingers can be linked in tandem
  → Allows recognition of longer, specific DNA sequences

What is the homeodomain DNA-binding motif and how does it recognize DNA?

• Homeodomain: conserved DNA-binding motif (~60 amino acids)

• Contains helix–turn–helix structure

• One helix = recognition helix

• Recognition helix inserts into the major groove

• Amino acid side chains form H-bonds with bases
  → Enables sequence-specific DNA binding (gene regulation

What is the leucine zipper DNA-binding motif and how does it bind DNA?

• Leucine zipper: motif with repeating Leu residues that form a coiled-coil dimer (dimerization)

• Two helices “zip” together → dimer formation

• DNA-binding region contains basic residues (Lys, Arg)

• These helices insert into the major groove

• Protein binds DNA by clamping onto both sides

→ Combines dimerization (Leu) + DNA recognition (Lys/Arg in major groove)

What is the helix–loop–helix (HLH) DNA-binding motif and how does it bind DNA?

• Consists of two α-helices connected by a loop

• One helix = recognition helix (binds DNA)

• Other helix = dimerization helix

• Amphipathic helices allow dimer formation

• Basic residues (Lys, Arg) mediate DNA binding

• Recognition helix inserts into the major groove

→ Combines dimerization + sequence-specific DNA binding

DNA molecules can be very long hence DNA must be BLANK to fit into cells

compacted

how does supercoiling relieve torsional strain in DNA?

• Separating DNA strands creates torsional strain (overwinding)

• DNA cannot freely rotate → strain builds up

• DNA relieves this by forming supercoils (writhe)

• Supercoiling converts twist → coiling in space
  → Lowers overall energy and relieves strain

AKA DNA supercoils to release twisting stress

polymerase does separates the strands during replication and introduces BLANK

Strain

Supercoiling is an intrinsic property of DNA’s BLANK structure

• supercoiling is an intrinsic property of DNA’s tertiary structure

• it occurs in all cellular DNAs and is highly regulated by each cell

what does it mean for DNA to be underwound?

fewer turns than relaxed DNA which creates torsional strain or less tightly twisted

• Relaxed DNA: ~10.5 bp/turn

• Underwound DNA: more bp/turn (e.g., ~12 bp/turn)

how does supercoiling stabilize underwound DNA?

• Underwound DNA is energetically strained

• DNA forms supercoils to compensate

• Converts underwinding (twist) → writhe (coils)
  → Stabilizes the molecule by lowering energy

Why is underwound DNA biologically useful?

• Facilitates strand separation

• Makes DNA easier to open

• Important for:

  • Replication

  • Transcription

What is the linking number (Lk) in DNA?

• Lk = number of times one DNA strand wraps around the other

• Applies to closed (circular) DNA

• Represents total helical turns
  → Topological property (cannot change without breaking DNA)

How are Lk, ΔLk, and superhelical density (σ) related in DNA?

• Lk = # base pairs ÷ bp per turn

  • Example: 2100 bp ÷ 10.5 = Lk₀ = 200 (relaxed DNA)

• ΔLk = Lk − Lk₀

  • Underwound example: ΔLk = −2 → Lk = 198

• σ (superhelical density) = ΔLk / Lk₀

  • Example: σ = −2 / 200 = −0.01

Key points:

• Nick (strand break) → Lk undefined

• Negative ΔLk = underwound DNA

underwound DNA is what kind of supercoil?

negative

overwound DNA is what kind of supercoil?

positive

what are topoisomers?

two forms of a circular DNA that differ only in a topological property such as linking number are referred to as topoisomers

What does topoisomerase I do?

• changes Lk in increments of 1

• cleaves one DNA strand

• relaxes positive and negative supercoils

• does NOT require ATP

• Example: Topoisomerase I

what does Topoisomerase II do?

• changes Lk in increments of 2

• cleaves both DNA strands

• relaxes positive and negative supercoils

• can introduce negative supercoils (prokaryotes)

• requires ATP (hydrolyzes ATP)

• examples: DNA gyrase, Topoisomerase II

How does DNA supercoiling affect migration in gel electrophoresis?

• Highly supercoiled DNA → more compact → travels faster (lower on gel)

• Relaxed DNA → more open/large → travels slower (higher on gel)

• Different bands = topoisomers (different Lk values)

• Topoisomerase I produces a ladder with ΔLk = 1 between bands

👉 Key idea: Shape (compact vs relaxed), not length, determines migration speed

What is the mechanism of Topoisomerase I?

• Binds DNA

• Cleaves one strand (via Tyr residue)

• Passes the uncut strand through the break

• Religates DNA
  → Relieves supercoiling without fully breaking the helix

How does Topoisomerase I change DNA topology?

• remove supercoils (especially negative supercoils)

• reduces torsional strain

• helps maintain DNA stability during replication/transcription

What is the mechanism of Topoisomerase I?

• Binds DNA

• Cleaves one strand (via Tyr residue)

• Passes the uncut strand through the break

• Religates DNA
  → Relieves supercoiling without fully breaking the helix

How does Topoisomerase I change DNA topology?

• Changes Lk in increments of 1

• ΔLk = ±1 per cycle

• Gradually relaxes supercoiled DNA
  → One “turn” removed at a time

• can remove negative supercoils

What is the function of Topoisomerase I?

• Removes supercoils (especially negative supercoils)

• Reduces torsional strain

• Helps maintain DNA stability during replication/transcription

TOPOISOMERASE I ALWAYS MOVES DNA TOWARD LK=0

draw topoisomerase I reaction

what is the mechanism of Topoisomerase II? what does it require?

• Binds two DNA double helices (segments)

• Cleaves both strands of one DNA segment

• Uses ATP (2 ATP hydrolyzed)

• Passes second DNA segment through the break

• Religates the cut DNA and releases strand

→ Changes Lk in increments of 2 (ΔLk = ±2)
→ Can relieve supercoils and introduce negative supercoils (prokaryotes)

what does topoisomerase III do in DNA decatenation?

• decatenates or separates interlinked circular DNA molecules

What is plectonemic supercoiling and what is its limitation?

• Plectonemic supercoiling: DNA coils around itself forming interwound helices

• Creates supercoil axis and branch points

• Common form of DNA supercoiling in cells

Limitation:

• Does NOT compact DNA enough for full cellular packaging
  → Additional structures (e.g., chromatin) are needed for higher-order compaction

this is an example of what kind of supercoiling and it is MORE or LESS compact?

• less compact and plectonemic

what kind of supercoiling is this?

• solenoidal and more compact — found in cells

What is a nucleosome and how does it package DNA?

• Nucleosome = DNA wrapped around a histone core

• Forms “beads on a string” structure
  → Basic unit of DNA packaging in eukaryotes

What is the role of histones and linker DNA in chromatin?

• Histones: proteins that compact DNA

• Linker DNA: stretches between nucleosomes

• Allows DNA to be organized and further compacted

👉 Key idea: Histones enable DNA to fit inside the cell while maintaining structure

histones are found in BLANK of all eukaryotic cells?

chromatin

what is histones main purpose as a protein?

• main purpose of histones is to allow DNA to wrap around it so it needs to have a surface that DNA likes

What is the nucleosome core particle? — not sure if high yield but just konw

• Core made of 8 histone proteins (histone octamer):

  • 2 × H2A, 2 × H2B, 2 × H3, 2 × H4

• ~146 bp of DNA wrapped around the histone core

• DNA wraps ~1.7 turns

• 146 bp of DNA wrapped around histone care

→ Fundamental unit of chromatin structure and DNA packaging

essentially nucleosome core particle is just DNA or 146 bp wrapped around a histone protein

What are histone tails?

• Amino-terminal (N-terminal) extensions of histone proteins

• Protrude outward from the nucleosome core

• Flexible and accessible outside the DNA-histone structure

“amino terminal tails of histone proteins that produce from the core particles. these tails are extensively post translationally modified and also paritpcien in DNA packaging“

What is the function of histone tails?

• Extensively post-translationally modified (e.g., acetylation, methylation)

• Help regulate DNA packaging (tight vs loose chromatin)

• use tails for information, to define where we are, and change information on tails

what are examples of histone modifications?

• methylation, phosphorylation, and acetylation

packaging of DNA into nucleosomes leads to BLANK?

supercoiling

How is DNA organized at a higher-order level beyond nucleosomes?

• DNA is organized into looped domains

• Loops are anchored to a protein scaffold (chromosomal scaffold)

• This scaffold becomes visible when histones are removed

• Helps compact DNA and organize chromosomes efficiently

• Key idea: DNA is not random → it is structured into loops for packing + regulation

how is DNA organized at a higher order level beyond nucleosomes?

• DNA is divided into active (euchromatin) and inactive (heterochromatin) compartments

• CTCF (binding protein) helps organize DNA into loops called TADs (Topologically Associated Domains)

• Active regions = loosely packed, transcriptionally active

• Inactive regions = tightly packed, transcriptionally silent (heterochromatin)

• Key idea: 3D DNA organization controls gene expression

long non coding RNAs and associated proteins also organize DNA in BLANK

chromosomes

BLANK AND BLANK organize DNA in the eukaryotic cell cycle

Cohesins and condensins

what is the difference between a semiconservative models vs a conservative model?

• semiconservative model: hybrid duplex of old and new strand —> RIGHT

• conservative model: duplex of only old or only newly synthesized DNA

DNA synthesis is performed by what enzyme?

DNA polymerases

DNA synthesis requires what and catalyzes what?

require:

• template strand to copy

• primer strand with 3’ OH

• dNTP substrates

catalyze:

• nucleophilic attack by 3’ OH

• phosphodiester bond formation

• 5’ —> 3’ synthesis

DNA synthesis is always X’ —> Y’

DNA synthesis is always 5’—> 3’

Why is base pair geometry important for DNA replication fidelity?

• DNA polymerase active site is shaped to fit correct pairs (A-T, G-C)

• correct pairs have proper geometry —> fit perfectly

• incorrect pairs have distorted shape —> don’t fit well

• this allows polymerase to select the right nucleotide

how accurate is DNA polymerase and why?

• Error rate: ~1 mistake per 10⁴–10⁵ nucleotides

• High fidelity comes from:

  • Base pair geometry recognition (shape-based selection)

  • Incorrect bases are rejected due to poor fit in active site

• Key idea: shape matters more than just hydrogen bonding

What is the role of 3’ → 5’ exonuclease activity in DNA replication?

• Provides proofreading function

• Removes incorrectly added nucleotides

• Works in the 3’ → 5’ direction

• Increases replication accuracy significantly

How does DNA polymerase correct a mistake?

• Incorrect base is added

• DNA is shifted to exonuclease site

• Wrong nucleotide is cleaved off

• DNA returns to polymerase site

• Correct nucleotide is added

DNA replication has three major stages what are they?

• initiation

• elongation

• termination

What is DUE? it typically has a bp region that is heavily concentrated with?

• DNA unwinding element

  • A-T rich region —> easier to separate strands (fewer H bonds)

  • site where DNA first unwinds

replication starts at specific sequences, where proteins bind and AT rich DNA unwinds first

what does the DnaA protein do?

• able to recognize the origin sequence

• recognizes the oriC sequence; opens duplex at specific sites in origin

what is the DnaB protein or helicase do?

unwinds the DNA

what does the DnaC protein do?

required for DnaB binding at the origin

How does DNA replication initiate at the origin (oriC) in bacteria?

1. DnaA binds origin (oriC):

   • DnaA-AAA+ binds specific sequences → wraps & bends DNA

2. DNA unwinding (DUE):

   • AT-rich DUE region melts → forms an open complex (bubble)

3. DNA bending proteins help:

   • Proteins like IHF assist in DNA deformation → promotes opening

4. Helicase loading:

   • DnaC loads DnaB helicase onto single-stranded DNA

5. Replication begins:

   • DnaB helicase further unwinds DNA → replication machinery assembles

what does initiation involve for DNA replication?

Initiation = protein binding → DNA bending → AT-rich unwinding → helicase loading → replication starts

what is priming in DNA replication and why is it necessary? what enzyme does it use?

• Primase synthesizes short RNA primers at the origin

• Provides a free 3′-OH group for DNA polymerase to start synthesis

• DNA polymerase cannot start de novo → must extend from a primer

• Primers are laid down on both strands as replication begins

How does DNA polymerization occur during replication, and in what direction is DNA synthesized? what enzyme does it utilize? DNA replication reads the leading strand from what?

• DNA polymerase III extends from RNA primers

• Synthesizes DNA only in the 5′ → 3′ direction

• Moves along the template strand reading 3′ → 5′

• Replication proceeds bidirectionally from the origin

• Both strands are copied simultaneously, but differently (leading vs lagging)