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Questions and Answers

What initiates the separation of double-stranded DNA during DNA replication?

Origin recognition complex (correct)

Topoisomerase

Single-stranded binding proteins

DNA polymerase

Which enzyme is responsible for unzipping the double-stranded DNA by breaking hydrogen bonds?

Ligase

Primase

Helicase (correct)

Topoisomerase

In eukaryotes, how many origins of replication (ori) are typically activated during DNA replication?

Hundreds

Dozens

Thousands (correct)

Millions

What is the rate of DNA synthesis in E. coli?

2000 nt/s Signup and view all the answers

What is the primary function of DNA ligase during DNA replication?

To join ribose-phosphate backbones Signup and view all the answers

Which statement about prokaryotic chromosomes is correct?

They have a single circular chromosome. Signup and view all the answers

During the bubble expansion in eukaryotic DNA replication, what is the first step?

Ori denatures along the chromosome. Signup and view all the answers

What role does topoisomerase play during DNA replication?

Relax supercoiling of double-stranded DNA Signup and view all the answers

What role does DNA polymerase III play in DNA replication?

It adds nucleotides to the 3' end of DNA. Signup and view all the answers

What is the primary function of primase in DNA replication?

To provide a starting point for DNA polymerase. Signup and view all the answers

What distinguishes the synthesis of the leading strand from the lagging strand during DNA replication?

Leading strand synthesis occurs continuously whereas lagging strand synthesis is discontinuous. Signup and view all the answers

Which of the following statements about Okazaki fragments is true?

They form on the lagging strand during DNA replication. Signup and view all the answers

Which action can DNA polymerase NOT perform?

Start a new DNA strand. Signup and view all the answers

What is the significance of DNA ligase in DNA replication?

It creates bonds between Okazaki fragments. Signup and view all the answers

What happens to the strands of DNA after replication?

Each new dsDNA contains one old strand and one newly synthesized strand. Signup and view all the answers

During DNA replication, how does DNA polymerase I function in relation to RNA primers?

It removes RNA primers and fills in gaps with DNA. Signup and view all the answers

What role do damage recognition complexes play in nucleotide excision repair?

They recognize damage beyond base mismatches. Signup and view all the answers

Which repair mechanism is specifically designed to address bulky lesions?

Nucleotide Excision Repair (NER) Signup and view all the answers

What is the purpose of the endonucleases in the nucleotide excision repair process?

To cleave phosphodiester bonds on either side of a lesion. Signup and view all the answers

Which protein complex is pivotal for separating DNA during nucleotide excision repair?

TFIIH complex Signup and view all the answers

How does the redundancy in DNA repair mechanisms contribute to genetic integrity?

It allows for multiple pathways to fix errors, reducing overall mistakes. Signup and view all the answers

What unique characteristic of DNA replication is observed at the ends of linear chromosomes?

It leaves a terminal gap that cannot be filled. Signup and view all the answers

What is the primary function of telomerase in relation to chromosomes?

To synthesize nucleotides at the chromosomal ends. Signup and view all the answers

Which of the following statements is true regarding telomeres?

They consist of a repeating motif of 5-8 nucleotides. Signup and view all the answers

What part does the accessory protein β-clamp play in DNA replication?

It helps DNA polymerase slide along single-stranded DNA. Signup and view all the answers

Which condition reflects DNA polymerase's efficiency in vivo compared to in vitro?

It is faster and more accurate in vivo. Signup and view all the answers

What is the role of the primase during telomerase activity?

To provide an RNA primer for DNA synthesis. Signup and view all the answers

What is meant by processivity in the context of DNA polymerases?

The number of nucleotides synthesized before the polymerase disassociates. Signup and view all the answers

How do DNA polymerases recognize the correct nucleotide to incorporate during DNA synthesis?

They rely on geometric fit and H-bond interactions. Signup and view all the answers

What is the primary function of DNA polymerases during DNA replication?

To insert nucleotides and proofread the sequence Signup and view all the answers

Which enzyme is responsible for recognizing chemically altered bases in base-excision repair?

Glycosylase Signup and view all the answers

How does DNA polymerase identify mismatched bases during proofreading?

It detects distortions in the double helix structure Signup and view all the answers

What role do methylated bases play in mismatch repair?

They help distinguish between old and newly synthesized DNA Signup and view all the answers

What happens during the base-excision repair process after the AP site is created?

The AP endonuclease cleaves the phosphodiester backbone Signup and view all the answers

Which protein is primarily involved in the initial recognition of mismatched nucleotides after replication?

MutS Signup and view all the answers

What type of repair mechanism utilizes multiple glycosylases specific to different bases?

Base-excision repair Signup and view all the answers

What occurs after MutH cuts the backbone of the newer DNA during mismatch repair?

DNA polymerase synthesizes a replacement for the excised region Signup and view all the answers

Study Notes

DNA Replication

DNA replication occurs during the S phase of the cell cycle to prepare chromosomes for either mitosis or meiosis.
DNA replication is a highly regulated process that ensures accurate duplication of the genome.
The process of DNA replication is similar to DNA repair mechanisms.
Initiation of replication begins at an origin of replication (ori), typically a short region rich in adenine (A) and thymine (T) bases.
Origin Recognition Complex (ORC) binds to the ori and promotes strand separation of double-stranded DNA (dsDNA).

Prokaryotic Replication

Most prokaryotes have a single circular chromosome.
Replication proceeds in a single replication bubble that expands bidirectionally until the two forks meet.
E. coli, a model organism, contains ~4.6 million base pairs in a single chromosome.
DNA synthesis occurs at a rate of ~2000 nucleotides per second (nt/s).
E. coli can replicate its entire genome in ~20 minutes.
Unlimited resources are a rare occurrence in nature, and using additional origins of replication in prokaryotes provides no benefit.

Eukaryotic Replication

Eukaryotic DNA synthesis is slower than in prokaryotes.
The human genome contains ~3.2 billion base pairs.
DNA synthesis rate is ~100 nts/s.
Eukaryotic chromosomes harbor thousands of origins of replication.
The mouse genome has ~100,000 potential origins, with approximately 20,000-30,000 activated during replication.
Simultaneous replication bubbles are present in the genome, speeding up the process.

Eukaryote Bubble Expansion

The origins of replication denature along the chromosome.
Replication bubbles form and expand bidirectionally.
Replication bubbles eventually meet.

Major Replication Machines

Origin Recognition Complex (ORC): a complex of initiator proteins that recognizes the origin of replication.
Helicase: responsible for unwinding dsDNA by breaking hydrogen bonds.
Single-Stranded Binding Proteins (SSB): stabilize single-stranded DNA (ssDNA) to prevent re-annealing.
Topoisomerase: relaxes supercoiling of dsDNA to prevent tension during unwinding.
Primase: synthesizes short RNA primers to initiate DNA synthesis, as DNA polymerase requires a pre-existing 3' hydroxyl group.
DNA Polymerase: catalyzes the synthesis of a new DNA strand by adding nucleotides to the 3' end of a pre-existing strand.
Ligase: joins the ribose-phosphate backbones of newly synthesized DNA fragments.

DNA Replication - Initiation

ORC binds to the ori, separating the DNA strands.
Helicase attaches to the replication fork and unwinds the dsDNA by breaking hydrogen bonds.
Topoisomerase relaxes the supercoiling of dsDNA that occurs ahead of the replication fork.
SSB proteins bind to ssDNA, preventing it from folding back on itself and re-pairing.

DNA Replication - 2

DNA Polymerase III is responsible for synthesizing the new DNA strands.
Primase (called DnaG in the figure) synthesizes a short RNA primer to provide a starting point for DNA polymerase III.
DNA polymerase III uses the 3' end of the RNA primer to add DNA nucleotides to the new strand.
Nucleotide addition occurs in the 5' to 3' direction.

DNA Polymerase Capabilities

Can: add nucleotides to the 3' end of a DNA strand, extend an existing strand.
Cannot: initiate a new strand, add nucleotides to the 5' end.
Deoxynucleotide triphosphates (dNTPs) provide the energy needed for DNA synthesis.

Leading/Lagging Strand Synthesis

Both strands in a dsDNA molecule are synthesized in the 5' to 3' direction.
However, because the strands are antiparallel, synthesis proceeds in opposite directions relative to the replication fork.
Leading strand: continuously synthesized in the direction of the replication fork.
Lagging strand: synthesized discontinuously in the opposite direction of the replication fork.

Okazaki Fragments

Discontinuous Okazaki fragments are synthesized on the lagging strand.
The direction of the template strand is the same as the direction of the replication fork.
DNA polymerase synthesizes in the opposite direction of bubble expansion, requiring it to continually start over.

DNA Replication - 3

DNA polymerase I removes the RNA primers, including those for Okazaki fragments.
The enzyme has 5' to 3' exonuclease activity.
RNA nucleotides are replaced by DNA nucleotides by DNA polymerase I.
DNA polymerase I cannot complete the backbone when reaching the next Okazaki fragment, leaving "nicks" in the lagging strand.

DNA Replication - 4

DNA polymerases can only add a new nucleotide to the 3' end of a strand.
This means they cannot directly join Okazaki fragments.
Ligase is responsible for recognizing "nicks" in the phosphate-deoxyribose backbone and creating a new deoxyribose-phosphate linkage, producing a continuous DNA molecule.

Basic Replication Summary

dsDNA is unwound into ssDNA.
DNA polymerase "reads" ssDNA templates in the 3' to 5' direction.
New DNA is synthesized in the 5' to 3' direction along each template strand.
Continuous DNA synthesis occurs along the leading strand in the direction of the replication fork.
Discontinuous DNA synthesis occurs on the lagging strand.

Semiconservative Replication

Each ssDNA strand serves as a template once the dsDNA molecule is separated.
After replication, each dsDNA molecule contains:
- one old piece of ssDNA
- one new piece of ssDNA

The Terminus Problem

DNA polymerase can synthesize the leading strand right to the end of the chromosomes.
The lagging strand, however, cannot be fully replicated because without a 3' hydroxyl group to begin synthesis, a terminal gap remains.
This gap increases with each replication, resulting in a 3' overhang at both ends of every linear chromosome.

Telomeres

Short tandem repeats (STRs) are found at the ends of linear chromosomes.
These repetitive sequences are typically 5-8 nucleotides long and vary between species.
They are non-coding, meaning they do not contain genes.
They function as protective caps for the ends of chromosomes, preventing loss of genetic material.
Human telomeric STRs are 10-15 kilobases long and consist of the repeating motif TTAGGG.

Telomerase Activity

Telomerase is an enzyme that extends telomeres.
It carries a small RNA molecule that is reverse complementary to the telomeric STR motif.
This RNA molecule acts as a primer for DNA synthesis by telomerase.
Telomerase binds to the 3' overhang and synthesizes a few nucleotides using its polymerase activity.
It then slides along the telomere, repeating the process and extending the telomere.

Telomerase Activity - 2

Once telomerase dissociates, primase synthesizes an RNA primer.
DNA polymerase uses this primer to synthesize DNA and fill the remaining gap.
Ligase seals remaining nicks in the backbone.

DNA Polymerase Efficiency

Processivity: the number of nucleotides synthesized before a DNA polymerase dissociates from the template strand.
Fidelity: the number of errors made during DNA synthesis.
DNA polymerases are capable of both high processivity and fidelity.
In vitro, DNA polymerase exhibits low efficiency, synthesizing only about 10 nucleotides before dissociating.

DNA Pol Processivity

In vivo, DNA polymerase utilizes the replisome to increase processivity.
The replisome is a complex of replication "machines" that function as a larger, coordinated system.
It includes the Pol III holoenzyme, which consists of leading/lagging strand polymerases, accessory proteins, and proteins that coordinate with helicase.
The replisome increases processivity by 1000 times.

Replisome

DNA polymerases on the leading and lagging strands form a dimer at the replication fork, which is linked by catalytic cores, increasing speed and reducing dissociation probability.
An accessory protein called the β-clamp acts like a ring that helps DNA polymerase slide along the ssDNA.
Other components, such as ligase, polymerase I, and topoisomerase, operate independently at different sites.

DNA Pol Cannot Read

DNA polymerase does not read the DNA sequence.
The incorporation of nucleotides is geometrically limited based on hydrogen bond interactions.
If a nucleotide fits in the active site, it is incorporated.
DNA polymerase is agnostic to the specific base differences between nucleotides.

DNA Pol Fidelity

DNA polymerases can mistakenly insert incorrect nucleotides.
They rely on proofreading to correct these errors.
3' to 5' exonuclease activity allows DNA polymerase to back up and remove mismatched nucleotides before they are incorporated into the growing strand.
This reduces the actual mutation rate significantly.

Pol Exonuclease Activity

Mismatched bases are detected by DNA polymerase.
1. The mismatch creates a lesion in the backbone causing a change in the polymerase's shape.
1. DNA polymerase backs up, removing one nucleotide in the 3' to 5' direction.
1. The mismatched nucleotide is removed.
1. Replication continues.

Base-Excision Repair (BER)

BER is the most important repair mechanism after polymerase proofreading.
Chemically altered bases are recognized by a specific glycosylase.
Glycosylases cleave the altered base, creating an apurinic/apyrimidic (AP) site, which lacks a base.
There are multiple glycosylases, each with a specific target base.

Base-Excision Repair - 2

An AP endonuclease recognizes the AP site and cleaves the phosphodiester backbone, creating a gap in the DNA backbone.
Another enzyme, dRpase, removes the remaining nucleotides.
This creates a stretch of missing ssDNA, enabling DNA polymerase to fill the gap.

Base-Excision Repair - 3

DNA polymerase inserts new nucleotides with the correct bases.
This is similar to the process during replication but does not require a primer.
Ligase seals the nick in the DNA backbone.
If the polymerase inserts a mismatched nucleotide, the process restarts.

Mismatch Repair

Mismatch repair occurs after replication to correct mismatched nucleotides.
MutS proteins recognize distortions in the double helix caused by mismatches and bind to the distorted site.
This triggers the replacement of the mismatched nucleotide.
For example: 5' G T T C 3' 3' T A A G 5'

Mismatch Repair - 2

Certain bases carry epigenetic markers.
Methylated adenines are found in prokaryotes.
Methylated cytosines are found in eukaryotes.
Methylation serves as a way to distinguish the parental strand from the newly synthesized strand, allowing repair mechanisms to target the incorrect nucleotide on the new strand.
MutS recruits other Mut proteins, which recognize the methylated base in the older DNA.
MutH cleaves the backbone of the newer DNA strand.

Mismatch Repair - 3

Helicase unwinds the DNA around the nicked site.
The newly synthesized DNA containing the mismatch is excised.
DNA polymerase synthesizes new DNA to fill the gap.
Ligase seals the gap left behind.

Nucleotide Excision Repair (NER)

NER is responsible for repairing bulky lesions in DNA that can result in gene dysfunction.
Damage recognition complexes detect damage beyond base mismatches.
There are two types:
- Global genomic NER (GG-NER) repairs damaged regions anywhere in the genome, typically when replication forks stall.
- Transcription coupled NER (TT-NER) is dedicated to repairing damaged sites in actively transcribed genes.

Nucleotide Excision Repair - 2

Damage recognition complexes recruit the TFIIH complex which contains two helicases.
These helicases separate the DNA on either side of the damaged region, which can span 2 to 100 nucleotides.

Nucleotide Excision Repair - 3

Endonucleases cleave the phosphodiester bonds on both sides of the lesion on one strand.
The damaged strand is removed.
DNA polymerase fills the gap using the undamaged strand as a template.
Ligase seals the gaps in the backbone.

DNA Repair Mechanisms

DNA repair mechanisms are highly redundant.
Without any repair mechanisms, the error rate is 1 in 100,000.
With polymerase proofreading, the error rate is 1 in 10,000,000.
With proofreading, BER, and MMR, the error rate is 1 in 1,000,000,000.
Each repair mechanism targets specific damage:
- BER repairs chemically altered bases.
- MMR corrects base mismatches.
- NER handles bulky lesions.

Replication & Repair Summary

Origins of replication
Replication machines
Leading/lagging strands
Telomerase
Replisome concept
Polymerase proofreading
Redundant repair mechanisms: BER, MMR, and NER

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