Cell Bio: DNA Replication and Repair

DNA replication

• process by which a copy of a DNA molecule is made

What phase does replication occur?

• interphase

Template

• a molecular structure that serves as a pattern for the production of other molecules

What enables DNA replication?

• base pairing

How is DNA replication semiconservative?

• each DNA molecule has an old strand and a new strand

Where does DNA replication begin?

• replication origin

“Initiator proteins” are required to start DNA replication. Where do the initiator proteins bind to pry the DNA apart?

• replication origin

Replication origin

• nucleotide sequence at which DNA replication is initiated

Which nucleotide sequence is the replication origin rich in?

• A-T

• easier to pry apart

Due to initiator proteins the double helix of DNA is opened and forms _____

• single stranded DNA templates

How many replication forks form at replication origins?

• 2

Replication fork

• Y shaped junction where DNA is being replicated

Eukaryotes have _____ replication origins

• multiple

What direction does replication move?

• opposite directions

DNA polymerase

• Enzyme that catalyzes the synthesis of a DNA molecule from a DNA template using deoxyribonucleoside triphosphate precursors

• adds nucleotides

During DNA replication a phosphodiester bond is formed between _____

• 3’ end of the growing DNA chain and the 5’ phosphate group of an incoming nucleotide

What direction is the new strand of DNA synthesized?

• 5’ to 3’

What do nucleotides form during DNA replication?

• Deoxyribonucleotides triphosphates (dNTPs)

What are the 4 types of dNTPs?

• dATP

• dGTP

• dTTP

• dCTP

dNTPs pair with bases of which strand?

• template

Does DNA polymerase require ATP to add new nucleotides?

• no

• gets energy from the breaking of the two terminal phosphates of dNTP

What direction does DNA polymerase read DNA?

• 3’ to 5’

Which end does DNA polymerase add nucleotides?

• 3’

DNA polymerase synthesizes the opposing strand in small fragments called _____

• Okazaki fragments

Which strand is made discontinuously via Okazaki fragments?

• lagging strand

The other strand not made of Okazaki fragments is the _____

• leading strand

How does DNA polymerase correct its mistakes?

• double checks to make sure it has the correct dNTP before adding it to the chain

• proofreading

Proofreading occurs at the same time as _____

• DNA synthesis

Can DNA polymerase start de novo (from nothing)?

• no

How does DNA polymerase get started?

• RNA primer

How is a RNA primer formed?

• Primase - an RNA polymerase that uses DNA as a template strand to produce a short RNA fragment that serves as the primer for DNA synthesis

Can primase start a strand of RNA de novo?

• yes

How many primers does the leading strand need?

• one RNA primer at the replication origin

How many primers does the lagging strand need?

• multiple

• because its synthe

What are the three additional enzymes required by the lagging strand?

• nuclease

• repair polymerase

• DNA ligase

Nuclease

• degrades RNA primers

Repair polymerase

• adds DNA where RNA primer used to be

DNA ligase

• seals backbone

What helps DNA polymerase access nucleotides ahead of replication forks?

• DNA helicase

• single strand DNA binding proteins

DNA helicase

• uses ATP to pry apart the two strands of DNA

Single strand DNA binding proteins

• latch on to the unpaired DNA strands to prevent them from repairing

What two things help DNA polymerase form the replication complex?

• clamp loader

• sliding clamp

clamp loader

• brings polymerases together (one on leading/lagging strand) and loops out a section of DNA that will be the lagging strand

Sliding clamp

• keeps polymerase attached to DNA

What enzyme helps to prevent supercoiling?

• DNA topoisomerase

How does DNA topoisomerase work?

• creates a single strand break then reseals the strand before helicase passes over

What are telomers?

• added copies of the same nucleotide sequence over and over at the end of the chromosome

Why are telomers needed?

• the lagging strand can’t be replicated to the end of chromosomes

• the chromosome gets shorter after each round of division

What enzyme replicates telomers?

• telomerase

Does telomerase require an RNA primer?

• no

• has it’s own RNA template

Is telomerase always active in cells?

• no

Cells that ____ quickly have higher expression of telomerase

• divide

Why is telomerase activity reduced after many rounds of division?

• safeguard against over proliferation of cells (Ex: cancer)

Depurination

• loss of purine base (A or G) due to water attack

Deamination

• cytosine becomes damaged and turns to uracil

thymine dimer

• covalent linkages between pyrimidines

• caused by UV radiation

• causes DNA to bulge

What is the basic repair pathway of DNA?

1. identify and remove damage

2. DNA polymerase fill in the missing nucleotides (5’ to 3’)

3. DNA ligase seals backbone

Base excision repair (BER)

• fixes small, non-helix distorting legions (EX: deaminated cytosine, depurinated bases)

Nucleotide excision repair (NER)

• removes bulky, helix distorting legions (Ex. thymine dimers from UV radiation)

Mismatch repair

• fixes replication errors that escape proofreading

• must distinguish between newly synthesized DNA from template strand

What mechanisms fix double strand breaks (DSB)?

• non-homologous end joining (NHEJ)

• Homologous recombination (HR)

NHEJ

• quick

• joins broken ends without a template

• leads to loss of nucleotides or mis joining

HR

• uses undamaged sister chromatid as a template

Steps of HR

1. nuclease chews damaged DNA

2. 3’ overhang invades homologous DNA sequence

3. DNA polymerase elongates invading strand

4. DNA ligase seals backbone