Ch 20: DNA Replication and Repair

What does it mean that DNA replication is semiconservative?

Each new DNA molecule has one old strand and one newly made strand.

What is the role of helicase in replication?

Helicase unwinds the DNA helix by breaking hydrogen bonds between base pairs using ATP.

Why does DNA replication need an RNA primer?

DNA polymerases can only add nucleotides to an existing 3′ OH group. Primase lays down an RNA primer to start the strand.

What enzyme synthesizes the RNA primer?

Primase

How is the leading strand synthesized?

Continuously in the 5′ → 3′ direction, following the replication fork.

How is the lagging strand synthesized?

Discontinuously in short segments called Okazaki fragments, each needing a new RNA primer.

What enzymes link Okazaki fragments together?

• DNA polymerase I replaces RNA primers with DNA

• DNA ligase seals the nicks between fragments

What increases the processivity of DNA polymerase?

The sliding clamp protein holds DNA polymerase to the template, allowing it to stay attached and replicate efficiently.

What ensures the accuracy of DNA replication?

• Base pairing rules

• Proofreading by DNA polymerase (3′ → 5′ exonuclease activity removes mismatched bases)

What is the role of DNA ligase?

Forms phosphodiester bonds between DNA fragments, sealing breaks in the sugar-phosphate backbone.

What is the role of endonuclease in replication?

Removes RNA primers or damaged bases from within the DNA strand (not just at ends).

Why can’t DNA polymerase replicate the 3′ ends of linear chromosomes?

DNA polymerase needs a primer and can only extend DNA in the 5′ → 3′ direction.
After removing the final RNA primer, there’s no upstream 3′ OH for DNA polymerase to fill in the gap, leaving a short unreplicated 3′ overhang.

What happens if telomeres aren't maintained?

Chromosomes would get shorter with each cell division, eventually leading to loss of essential genes and cell death or aging.

What are telomeres made of?

Repeating non-coding DNA sequences (e.g., TTAGGG in humans) at the ends of chromosomes that protect genetic material.

What is telomerase and what does it do?

Telomerase is an RNA-dependent DNA polymerase.
It uses its own RNA template to add repeats to the 3′ end of DNA, extending telomeres.

What is the role of telomerase RNA?

Acts as a built-in template to extend telomere sequences (e.g., it contains an AAUCCC sequence to guide TTAGGG addition in humans).

In which cells is telomerase active?

• Stem cells

• Germ cells

• Cancer cells
  It’s inactive or very low in most somatic cells.

How is telomerase linked to cancer?

High telomerase activity allows cancer cells to keep dividing without losing DNA, giving them immortality.

What are common causes of DNA damage?

• UV light (causes thymine dimers)

• X-rays / radiation (causes strand breaks)

• Chemical mutagens (like alkylating agents)

• Replication errors

What is direct repair?

A repair mechanism that reverses damage without cutting DNA.
Example: Photolyase breaks UV-induced thymine dimers using light.

What is base excision repair (BER)?

Fixes small, non-bulky lesions (e.g., deaminated bases or oxidized bases):

1. DNA glycosylase removes damaged base

2. AP endonuclease cuts backbone

3. DNA polymerase + ligase fill and seal the gap

What is nucleotide excision repair (NER)?

Fixes bulky lesions like thymine dimers or chemical adducts:

1. Enzymes cut a section of damaged strand

2. DNA polymerase fills the gap

3. DNA ligase seals it

What is mismatch repair (MMR)?

Fixes errors missed by proofreading:

1. Detects mismatch

2. Removes stretch of new strand

3. DNA polymerase + ligase repair it

What is nonhomologous end joining (NHEJ)?

Repairs double-strand breaks by directly joining DNA ends.
Fast but error-prone (may delete bases).

What is homologous recombination repair?

Uses a sister chromatid as a template to fix double-strand breaks.
Very accurate, occurs after DNA replication.

Why can DNA repair sometimes cause mutations?

Some repair systems (like NHEJ) can introduce errors or deletions, especially when no template is available.

What is DNA supercoiling?

DNA is overwound or underwound, creating torsional stress.
Supercoiling helps compact DNA and affects access to genes.

What causes DNA supercoiling?

Helicase activity during replication or transcription unwinds DNA, which can lead to over- or underwinding of nearby regions.

What are the two types of supercoiling?

• Positive supercoiling = overwinding

• Negative supercoiling = underwinding (makes DNA easier to unwind)

What do topoisomerases do?

They relieve supercoiling by cutting DNA, twisting or untwisting it, and then resealing the break.

What’s the difference between Topoisomerase I and II?

• Topo I: Cuts one strand, no ATP needed

• Topo II (like DNA gyrase): Cuts both strands, uses ATP

How is DNA packaged in eukaryotes?

DNA wraps around histone proteins to form nucleosomes (like beads on a string)

What are nucleosomes?

Units of chromatin with DNA wrapped around 8 histone proteins (2 each of H2A, H2B, H3, and H4)

What is higher-order chromatin structure?

Nucleosomes fold into 30 nm fibers, which coil and loop into chromosomes during mitosis.

What do restriction enzymes (endonucleases) do?

They cut DNA at specific sequences, usually palindromes.
Used to create sticky or blunt ends for cloning.

What is DNA ligase used for in molecular biology?

Joins DNA fragments by forming phosphodiester bonds between the sugar-phosphate backbones.
Essential for making recombinant DNA.

What is a vector in DNA cloning?

A DNA molecule (like a plasmid) used to carry and replicate foreign DNA in a host organism.

What features do plasmid vectors have?

• Origin of replication

• Selectable marker (like antibiotic resistance)

• Multiple cloning site (MCS) with restriction sites

What is PCR (polymerase chain reaction)?

A method to amplify a specific DNA sequence using repeated cycles of:

1. Denaturation

2. Annealing (primers bind)

3. Extension (DNA synthesis by Taq polymerase)

What enzyme is used in PCR?

Taq polymerase, a heat-stable DNA polymerase from Thermus aquaticus.

How is DNA sequencing done (Sanger method)?

• Uses DNA polymerase and dideoxynucleotides (ddNTPs)

• ddNTPs stop chain elongation

• Results in DNA fragments of varying lengths that are read by a sequencer

What is a ddNTP and why is it used in sequencing?

A dideoxynucleotide lacks a 3′ OH, so it stops DNA strand growth.
Used to label and terminate DNA strands in sequencing.