Molecular Biology Quiz: DNA Ligase and Enzymes

Optional Textbook Reading

• Chapter 24, pages 891-892, 895-896
• Chapter 25, pages 914-929
• Chapter 26, page 993

Learning Objectives

• Describe the central dogma of molecular biology
• Interpret experimental data that helped determine DNA function and structure
• Describe the structure of DNA, RNA, and genomes in prokaryotic and eukaryotic cells
• Identify DNA replication proteins and their functions
• Predict DNA sequence based on the template strand used for synthesis
• Differentiate various mechanisms for ensuring genomic accuracy
• Diagram replication forks and DNA polarity
• Describe the importance of telomeres and their replication mechanisms

Supplemental Resources

• Supplemental videos and practice problems on Canvas
• Discussion hours: Mondays, Tuesdays, and Fridays, 2-3 PM in the auditorium
• Zoom "Coffee & Review" hour: Thursdays, 8-9 AM
• Campuswire
• One-on-one meetings (groups of up to 5 welcome): Canvas

Lecture Outline

• Enzymes that process the genome
• DNA replication is semi-conservative
• DNA polymerases
• Prokaryotic DNA Replication
• Eukaryotic DNA Replication

The "Central Dogma" of Molecular Biology

• DNA → RNA → Protein
• Transcription: DNA to RNA
• Replication: Duplicating the genome
• Translation: RNA to protein

Enzymes that Process the Genome

• General groups of enzymes processing genomes will be discussed
• At least one specific example of each enzyme class explored in the video will be given
• Track the family each example belongs to

Polynucleotide Synthesis

• Synthesis always 5' → 3'
• Uses nucleoside triphosphates
• Nucleotide selection based on base pairing with template
• DNA polymerase adds deoxyribonucleotides to the 3' end of a DNA chain
• RNA polymerase adds ribonucleotides to the 3' end of an RNA chain
• Reverse transcriptase adds deoxyribonucleotides to a DNA chain using an RNA template

Nucleases Digest Polynucleotide Chains

• DNase is specific for DNA, RNase for RNA
• Exonuclease breaks a phosphodiester bond at a polynucleotide chain end (either 5' → 3' or 3' → 5')
• Endonuclease breaks a phosphodiester bond within a polynucleotide chain (can be sequence-independent or -specific, single or double strand break)
• Excinuclease breaks two phosphodiester bonds within a single polynucleotide chain

Restriction Endonucleases/Enzymes

• Type of endonuclease, only breaking phosphodiester bonds at specific sequences (restriction sites)
• Sequences are short, palindromic, and 4-6 base pairs long
• Palindromes read the same on both strands in 5' → 3' direction

DNA Ligase

• Links two existing DNA chains by forming a phosphodiester linkage
• Joins continuous 5' → 3' DNA strands
• In vivo, primarily seals single-strand breaks (nicks)

Recombinant DNA Technology

• DNA processing enzymes create new combinations of sequences (recombinant DNA)
• In biotechnology, DNA ligase joins DNA molecules end-to-end (blunt ends or compatible sticky ends)
• Endonucleases create sticky ends

iClicker Question #2

• Identify the enzyme catalyzing the given reaction (5'→ 3' exonuclease)

DNA Replication Is Semi-Conservative

• Drawing of relevant experiments/processes will enhance learning
• Teaching others is an effective way to learn.

What is the Mechanism of DNA Replication?

• Conservative replication yields one original DNA and one entirely new DNA molecule.
• Semi-conservative replication yields two DNA molecules; each has one parental strand and one new strand
• Dispersive replication yields two mixed DNA molecules (parental/new)

Meselson and Stahl Experiment

• Study of DNA replication in E. coli
• Used isotopes of nitrogen to distinguish newly synthesized DNA from the original
• Demonstrated semi-conservative replication

DNA Replication is Semi-Conservative (Page 14)

• Diagram illustrating semi-conservative replication

DNA Polymerases

• DNA polymerases use Roman numerals for bacterial and Greek letters for eukaryotes

DNA Polymerase Catalyzes DNA Synthesis

• Catalyzes synthesis of one nucleoside monophosphate at a time
• Always in 5' → 3' direction
• Requires DNA template strand, RNA primer, and deoxyribonucleoside triphosphates (dNTPs)
• Catalytic site is located in the "palm" of the enzyme

Mechanism of DNA Polymerase

• Catalyzes extension of a DNA strand, one dNTP at a time
• Synthesizes in 5' → 3' direction, requires template and primer
• Phosphoryl group transfer reaction involving Mg2+ ions and 3'-OH of the 3' nucleotide
• Releases pyrophosphate (PPi)

Replication is Accurate

• Errors during DNA replication create mutations.
• The average E. coli mutation rate is ~1 bp per 109-1010 bp
• DNA Pol active site restricts base pairing (Watson-Crick-Franklin)
• Accuracy of ~10⁻⁴ - 10⁻⁵

Proofreading

• High-fidelity DNA polymerases have two active sites:
• A catalytic site for synthesis
• A 3ʼ→5' exonuclease site for removing mismatched nucleotides

Prokaryotic DNA Polymerases

• Table with different types of prokaryotic DNA polymerases , structural genes, subunits, 3'→5' exonuclease function, 5'→3' exonuclease function, polymerization rate, and processivity, cellular role .

IV. Prokaryotic DNA Replication

• Replication "bubbles" / "forks" must be drawn to illustrate all 5' and 3' ends, replication fork movement, origin, and locations of all enzymes
• This is important to fully understand multiple concepts

DNA Synthesis Is Bidirectional

• Synthesis begins at an origin of replication
• Occurs in both directions from origin
• Synthesis occurs at replication forks

Replication Fork

• Where parent DNA is used as a template for replication
• Leading strand synthesized continuously
• Lagging strand synthesized discontinuously.

"Firing" of an Origin is Tightly Regulated

• Replication occurs only once per cell cycle in prokaryotes
• Prokaryotic DNA is haploid with two copies after replication.
• Initiation is a key regulated step controlling DNA synthesis

Bacterial DNA Replication Initiation Occurs at OriC

• Unique 245 bp sequence
• R1-5 and 11-3 are binding sites for DnaA protein
• DNA unwinding element (DUE) is an AT rich segment where strand separation occurs
• IHF and FIS are binding sites for replication initiation factors

Steps of E. coli DNA Replication Initiation

• DnaA binds ATP to become active
• DnaA proteins bind to oriC and cause DNA denaturation at the DUE (DNA unwinding element)
• DnaC loads DnaB helicase at both replication forks
• Helicase unwinds DNA, leads the replication fork separation
• DnaC dissociates
• DNA polymerase and additional proteins are added to both DnaB helicases
• Hydrolysis of ATP releases DnaA.

DNA Methylation Regulates Initiation

• oriC DNA is methylated by Dam methylase
• Methylates the N(4) position of A in GATC sequences
• After replication, DNA is hemimethylated
• Hemimethylated oriC associates with the plasma membrane
• Replication can restart when the DNA fully methylates again

DNA Polymerase III Holoenzyme

• Core polymerase catalyzes DNA synthesis
• Clamp loader serves as a scaffold for DNA polymerase III complex
• Assembles the β clamp onto DNA using ATP
• Coordinates replication fork by interacting with helicase.

Primase Generates Primers for Pol III

• DNA polymerase III requires a primed DNA template
• Primase is an RNA polymerase. It is template dependent and primer independent.
• Synthesizes small RNA primers at the start of the leading strand and each Okazaki fragment.
• Primer synthesis starts at either CTG or CAG

Topoisomerases

• Enzymes that add or remove supercoils in DNA to relieve torsional stress from unwinding or overwinding DNA
• Prevents DNA damage from supercoiling

Topoisomerase Type I

• Breaks one phosphodiester bond (nick)
• Passes the intact strand through the break
• Seals the nick
• Relaxed DNA

Topoisomerase Type II

• Breaks one phosphodiester bond in each strand (double strand break)
• Passes an intact segment of double-stranded DNA through the break
• Reseals the break, relieving positive supercoils

iClicker Question #1

• Identify the protein recognizing and binding to the oriC sequence (in E. coli) during the replication's initiation process. (DnaA)

The Prokaryotic Replisome

• DNA gyrase removes positive supercoils ahead of the replication fork
• Single-strand binding protein (SSB) protects single-stranded DNA from nucleases
• Holoenzyme synthesizes both strands simultaneously.

Lagging Strand Synthesis Part I

• DnaB helicase unwinds DNA along the lagging strand template in the 5' → 3' direction
• Primase (DnaG) associates with helicase and synthesizes short RNA primers
• Primase activity is at replication fork for Okazaki fragments.
• A new β clamp is loaded onto the lagging strand at each new RNA primer through the use of the clamp loader.

Lagging Strand Synthesis Part II

• New β clamp loading at each new RNA primer on the lagging strand through clamp loader
• Clamp loader binding ATP
• Clamp loader opening the β clamp due to ATP hydrolysis

Lagging Strand Synthesis Part III

• DNA Pol III pauses when it reaches the previous Okazaki primer
• Pol III releases the β sliding clamp and transfers to the next primer
• Lagging-strand core polymerase pauses, releases its β clamp, is then transferred to a newly loaded β clamp
• Newly loaded β clamp is used to complete lagging strand synthesis, DNA repair.

Lagging Strand Synthesis Part IV

• Lagging-strand core polymerase initiates the synthesis of the next Okazaki fragment.
• Clamp loader acquires a new β clamp in preparation for loading onto the next primer.
• "Watch DNA Replication In Action" supplemental video

Lagging Strand Processing

• DNA Pol III pauses at previous Okazaki primer after synthesis completes
• Pol III releases its β sliding clamp and transfers to a newly loaded clamp for the next fragment
• Exonuclease removes the RNA primer
• DNA Pol I synthesizes the DNA filling the gap
• DNA ligase repairs the nick in the backbone to join the Okazaki fragments

Replication of a Circular DNA Molecule

• Bidirectional, semi-conservative replication yields two identical DNA molecules
• After replication, circular chromosomes are linked together (catenated)

Separation of Catenanes by Topoisomerase IV

• Catenanes are topologically interlinked circular chromosomes
• Separation involves Topoisomerase IV, a type II topoisomerase, to separate DNA circles

iClicker Question #2

• What enzyme is considered faulty if DNA strands aren’t denaturing during the replication process? (Helicase (DnaB protein))

V. Eukaryotic DNA Replication

• Study tip: When completing practice problems, ensure that you can explain why the correct answer is correct AND why each incorrect answer is incorrect. If you are not able to explain why an incorrect answer is wrong, you need to review the information.

Prokaryotic & Eukaryotic DNA Replication Are Similar

• Many DNA polymerases in eukaryotes (approx. 15) with specialized functions
• Other DNA polymerases involved in various functions (DNA repair)
• DNA synthesis is semi-conservative and bidirectional
• Mechanism is template- and primer-dependent
• DNA synthesis occurs in the 5' → 3' direction
• Continuous leading strand synthesis
• Discontinuous lagging strand synthesis
• Some DNA polymerases are high-fidelity (have proofreading)

Unique Features of Eukaryotic DNA Replication

• Eukaryotic chromosomes can be much longer than bacterial DNA and linear
• Replicase has both DNA Pol α and DNA Pol ε
• Primase is in a complex with DNA Pol α
• Replication initiation is different in eukaryotes (multiple origins of replication)
• Coordinate regulation of origins (requires “licensing”)
• Replication elongation, rate, and Okazaki fragment size similar but not identical compared to prokaryotes
• Removal of RNA primers and replacement with DNA is different in eukaryotes
• Replication of telomeres unique to eukaryotes

The Eukaryotic Cell Cycle

• Cell division occurs in 4 stages (G1, S, G2, and M phases)
• Regulation involves cyclins and cyclin-dependent kinases (CDKs)

Multiple Origins of Replication

• Humans have 46 chromosomes and have at least 46 replication origins
• DNA synthesis rate is ≈ 50 nucleotides per second; chromosome 1 has ~2.46 x 108 base pairs.
• It would take ~1 month to replicate just chromosome 1 if there were only one replication origin

Licensing Coordinates Replication Initiation

• Origin replication complexes (ORC) bind tightly to DNA in G1 phase.
• Cell division cycle 6 (CDC6) and CDT1 join and load helicase (MCM) to the origin.
• MCM translocates 5' → 3' along the leading strand template using ATP
• Replication is initiated in S phase via phosphorylation of complex proteins by cyclin-CDK.
• Replisome assembled, bidirectional synthesis initiated

The Eukaryotic Replisome

• MCM is the helicase used in leading strand synthesis.
• High processivity DNA polymerase: epsilon used for leading strand synthesis.
• Proofreading delta used for lagging strand synthesis.
• DNA Pol alpha-primase contains primase for RNA primer synthesis and a separate DNA synthesis activity.
• Replication factor C(RFC) is the clamp loader
• Proliferating cell nuclear antigen (PCNA) is the sliding clamp
• Replication protein A (RPA) is a single-strand DNA binding protein (SSB)

Lagging Strand Processing

• Replication elongation essentially the same as seen in prokaryotes; one difference: replisome uses two different core enzymes.
• Another big difference is in the completion of the lagging strand, which is accomplished by (strand-displacing) DNA polymerase ε
• Flap endonuclease-1 (FEN1) clips off the overhang
• DNA ligase repairs the nick in the backbone to join the Okazaki fragments

Synthesis of Linear Chromosomes

• Humans have 46 linear chromosomes
• Replication of ends poses problems - they are primers for DNA polymerase
• Telomeres are repeated DNA at the end of chromosomes that protect them
• Due to this lack of a template, telomeres shorten with each replication cycle, and genes may be lost

Telomeres

• DNA structures at the ends of eukaryotic chromosomes
• Consist of a repetitive short sequence (e.g., TTAGGG in humans)
• TG strand is longer than the complementary CA strand.
• No genes are within the telomeres;
• Telomeres shorten each replication cycle; If telomeres shorten too much, genes are lost

T Loops in Telomeres

• Specialised structure that sequesters the single-stranded end of the telomere through base pairing
• Protects 3' end of chromosomes from nucleases and enzymes repairing double-strand breaks
• Proteins are bound to form the T loop
• Shelterin proteins protect the single-stranded 3' end within a DNA duplex.
• TRF1 and TRF2 bind looped DNA, TTGGGG repeat factor.

Electron micrograph of T Loop

• Image of a T loop

Telomere Length Shortens with Age

• Shortening of telomeres in somatic cells reflects cellular aging.
• Critical telomere length may trigger cell-cycle arrest or programmed cell death.
• Rapid aging phenotypes in diseases like progeria.
• Germ and stem cells don’t undergo telomere shortening

Telomere Synthesis

• Telomerase is specialized reverse transcriptase:
• Ribonucleoprotein containing RNA + protein
• Contains an internal RNA segment for template-based synthesis of the telomere TG strand
• Telomerase synthesis of TG strands extends the telomere.
• Complimentary CA strands synthesized separately later.
• The process repeats many times

Telomerase is Expressed in Some Cancers

• Sperm, eggs, and embryonic stem cells make telomerase.
• Somatic cells do not produce telomerase.
• Many cancer cells express telomerase.
• ALT (Alternative Lengthening of Telomeres).
• Cancer is uncontrolled cellular growth.

Test your knowledge on the role of DNA ligase and various enzymes involved in DNA replication and synthesis. This quiz covers fundamental concepts in molecular biology, including the functions of DNA polymerases and restriction endonucleases. Perfect for students looking to assess their understanding of enzymatic processes in genetics.