Molecular Biology BIOL-4310 11

Is DNA replication conservative or semiconservative?

semiconservative [one strand old material and other strand newly generated]

What direction does DNA polymerase extend DNA?

5’ to 3’

What is the direction of replication fork movement?

5’ to 3’

What is the direction of synthesis in DNA replication?

3’ to 5’

Leading Strand

Strand synthesized continuously in the direction of replication fork movement

Lagging Strand

Strand synthesized discontinuously in opposite direction [of direction of replication fork movement]. Consists of RNA Primers and Okazaki fragments joined together by ligase

Where is DNA replication initiated?

Origins of replication

What does it mean when it is said that DNA Replication is semi-continuous?

One strand is made continuous and the other is made discontinuous

How does DNA replication continue after initiation?

Proceeds bidirectionally from the origin, at growing points known as replication forks

What does DNA Polymerase and beta-clamp do in DNA replication?

DNA Polymerase main enzyme that replicated DNA

beta-clamp holds Polymerase onto DNA strands

What does Helicase do in DNA replication?

Unwinds DNA [Uses ATP to do so]

What does Topoisomerase do in DNA replication?

Untwist helical DNA

What does Primase do in DNA replication?

Synthesizes RNA primers

What does Ligase do in DNA replication?

Seals pieces of DNA together/Okazaki Fragments

What does SSB [Single-Strand Binding Proteins] do in DNA replication?

Prevents DNA strands from reannealing with each other

How many origins of replication are in prokaryotes and eukaryotes?

Pro: 1

Euk: Multiple

What does DNA polymerase need to synthesize DNA?

A primer and template strand

What does DNA Polymerase do to extend new strand of DNA?

adds dNTPs to 3’ end of a primer

What happens when dNTP binds with DNA Polymerase?

Creates a specific active site, where only correct base pairs fit

How is dNTP incorporated into the template strand?

When dNTP correctly base pairs with the template, pyrophosphate is released

How accurate is DNA Polymerase?

Very accurate [High fidelity]

What happens if DNA Polymerase detects incorrect/mismatched base pairs?

3’ to 5’ exonuclease activity is used to remove incorrect nucleotide/ proofreading

What does the structure of DNA Polymerase resemble?

A hand

What happens to the structure of DNA Polymerase when a correct nucleotide is added?

The “fingers” close around the strand very quickly

Mutation

Change in DNA sequence

How often does DNA Polymerase make mistakes?

1 error every 10^4-10^5 nucleotides copied

How does proofreading improve DNA Polymerase error rate?

by 10²-10³, 10^9-10^10 overall

What does a mutation consist of?

Conversion through replication into an incorrect base pair to create a stable, inheritable mutation

What counteracts mutations?

DNA repair mechanisms

What is an issue with DNA repair mechanisms?

Typically energy intensive

What are most mutations classified as?

deleterious

Where do mutations occur that can lead to cancer?

mutations in oncogenes and tumor suppressor genes that control cell division cycle

Point Mutation

Change in a single base pair

What are the types of point mutations?

-Silent

-Nonsense

-Missense

Silent Mutation

Codes for the same amino acid [ex. TTC→TTT Bot code for Lys]

Missense Mutation

Results in a different amino acid

Nonsense Mutation

Changes amino acid to a STOP codon

Conservative Missense

Changes to a chemically similar Amino acid [ex. Lys→Thr]

Nonconservative Missense

Changes to a chemically different amino acid [ex. Lys→Thr]

How many steps are most mutations produced in?

2 steps

Indel Mutations

Insertion or deletion mutation. gain [insertion] or loss [deletion] of one or more base pairs

Frameshift Mutation

Altered amino acid sequence beginning at the mutated codon, changes the protein reading frame

What kind of Indel mutations preserve the reading frame?

Where multiples of three base pairs are inserted/deleted

What can produce abnormal chromosomes?

Large-scale mutations such as deletions, duplications, inversions, or exchanges [translocations], or insertions

What can be formed from large-scale mutations?

Fused genes, which can cause cancer

What are the types of single chromosome mutations?

-Deletion

-Duplication

-Inversion

What are the types of multiple chromosome mutations?

-Insertion

-Translocation

Deletion

Loss of DNA sequences, truncating the chromosome

Duplication

DNA sequence is copied, elongating chromosome

Inversion

DNA segment is inverted/flipped 180 degrees

Insertion

A DNA sequence is removed from one chromosome and placed into another one

Translocation

Chromosomes “trade” DNA sequences

What is used to test the mutagenicity of a compound?

The Ames Test

Mutagen

Agent that causes mutations

Transition Mutation

Change of base pair from purine→purine or pyrimidine→pyrimidine

Transversion Mutation

Change of base pair from purine→pyrimidine or pyrimidine→purine

Trinucleotide repeat diseases

When a repeated 3 nucleotide sequence is expanded to a point where the abnormal number of repeats causes disease. Disease caused by abnormal amount of repeats

What does the Ames Test Consist of?

-Uses Salmonella that carries mutations in gene needed for histidine synthesis

-Liver enzymes are added to potential mutagen

-Mixture is added to filter paper disk

-Mutated Salmonella added to two dishes, one empty and the other with the paper disk

-If bacteria grows around paper disk, the potential mutagen can cause mutations