Biochem Lecture 8a

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Questions and Answers

What is the main consequence of alterations in the structure of nucleotides and nucleic acid?

Formation of mutations in the DNA sequence, which can be linked to cancer (correct)

Enhanced stability of the DNA structure

Reduced mutagenesis

Increased replication efficiency in DNA synthesis

In DNA repair, what is the role of the undamaged strand?

It increases the rate of mutations

It promotes DNA replication errors

It serves as a template for correcting the majority of damaged strands (correct)

It is damaged further during the repair process

What happens when some base changes escape repair mechanisms?

No changes occur in the daughter DNA

An incorrect base becomes a template in replication (correct)

The original DNA sequence is restored

DNA repair proteins prevent any errors

How is the accumulation of mutations in eukaryotic cells related to cancer risk?

Strong correlation between mutation accumulation and cancer development Signup and view all the answers

What percentage of lesions become actual mutations due to DNA repair mechanisms?

1/1000 Signup and view all the answers

What is the main consequence of unrepaired lesions in DNA?

Development of mutations, which can lead to cancer Signup and view all the answers

In the AMES test, what does the presence of colonies in a His-free medium indicate?

The compound mutated the Salmonella, restoring the ability to synthesize His. Signup and view all the answers

What is the purpose of the AMES test developed by Bruce Ames?

To indicate the mutagenic potential of a compound. Signup and view all the answers

What is the significance of the clear areas around the filter paper in the AMES test for carcinogens?

They indicate where mutagen concentration is lethal to cells. Signup and view all the answers

What does the growth of few small colonies of S.typhimurium on a histidine-free medium indicate?

Spontaneous back-mutations have occurred allowing histidine biosynthesis to operate. Signup and view all the answers

How does a mutagen affect the number of colonies in the AMES test?

It greatly increases the rate of backmutation and hence the number of colonies. Signup and view all the answers

What enzyme does a mutation in Salmonella strain used in the AMES test affect?

Enzyme involved in histidine biosynthetic pathway. Signup and view all the answers

Which protein binds specifically to a mismatched base pair in both bacteria and eukaryotic cells?

MutS Signup and view all the answers

In E. coli, which protein is responsible for nicking unmethylated GATC sequences to initiate the mismatch repair process?

MutH Signup and view all the answers

What enzyme is responsible for filling the resulting gap with DNA in the completion of methyl-directed mismatch repair in E. coli?

DNA polymerase III Signup and view all the answers

What protein triggers the degradation of the nicked strand through the mismatch in eukaryotes?

MutL Signup and view all the answers

Which protein is responsible for scanning the nearby DNA for a nick in both bacteria and eukaryotic cells?

MutL Signup and view all the answers

Which protein removes a segment of the new strand between the MutH cleavage site and a point just beyond the mismatch in E. coli?

Exonuclease Signup and view all the answers

What is the role of DNA Pol I in repairing DNA in the AP endonuclease repair process?

Synthesizes new DNA Signup and view all the answers

In the process of base-excision repair, what is the function of DNA glycosylase?

Recognizes a damaged base and cleaves between the base and deoxyribose Signup and view all the answers

What is the primary role of an AP endonuclease in DNA repair processes?

Cleaves the phosphodiester backbone near the AP site Signup and view all the answers

Which enzyme is responsible for sealing the nick after DNA Pol I dissociates in base-excision repair?

DNA ligase Signup and view all the answers

What activity of DNA Pol I is crucial during the repair synthesis in base-excision repair?

5′→3′ exonuclease activity Signup and view all the answers

What is the initial recognition step for damaged bases in base-excision repair?

Recognition by a DNA glycosylase Signup and view all the answers

Which enzyme performs nicktranslation activity during DNA repair processes?

DNA Pol I Signup and view all the answers

What is the function of MutL in eukaryotes?

Scans DNA for a nick and aids in the removal of the damaged strand Signup and view all the answers

What is the main function of DNA glycosylases in base excision repair?

Recognize specific lesions and cleave N-glycosidic bond Signup and view all the answers

Why is the removal of uracil from DNA by uracil glycosylase important?

Cytosine spontaneously deaminates to Uracil Signup and view all the answers

What is the significance of other glycosylases creating AP sites in DNA?

To signal for further repair processes Signup and view all the answers

Which protein scans the DNA for mismatches by testing for sites that can be readily kinked?

MutS protein Signup and view all the answers

What is the role of MutS protein in eukaryotic cells?

Scanning the DNA for mismatches by testing kinked sites Signup and view all the answers

Study Notes

DNA Repair and Mutations

DNA damage can lead to mutations, which are linked to cancer, and can occur through chemical reactions and physical processes that alter nucleotide and nucleic acid structure.
Most DNA damage is corrected using the undamaged strand as a template, but some base changes can escape repair and lead to mutations.
The accumulation of mutations in eukaryotic cells is strongly correlated with cancer, and most mutagens are also carcinogens.
The human genome contains genes for over 130 repair proteins.

Vocabulary of DNA Lesions

A lesion is a type of DNA damage that can become a mutation if left unrepaired.
Mutations can be substitutions (point mutations), deletions, or additions.
A silent mutation has almost no effect on gene function.

The Ames Test

The Ames test is a method used to determine the mutagenic potential of a compound.
It uses a strain of Salmonella with a mutation that makes it unable to synthesize histidine.
The test involves adding a compound to a plate of Salmonella and seeing if it grows in a histidine-free medium.
Colonies that grow indicate that the compound has mutated the Salmonella, restoring its ability to synthesize histidine.

DNA Repair Mechanisms

Methyl-Directed Mismatch Repair in E. Coli

The combined action of DNA helicase II, SSB, and one of four different exonucleases removes a segment of the new strand between the MutH cleavage site and a point just beyond the mismatch.
The resulting gap is filled in by DNA polymerase III, and the nick is sealed by DNA ligase.

Nick-Directed Mismatch Repair in Eukaryotes

In eukaryotes, the interaction between MutS:MutL complex and replication processivity factor (bclamp) activates the latent MutL endonuclease to nick the error-containing daughter strand.
The MutS protein binds specifically to a mismatched base pair, while MutL scans the nearby DNA for a nick.
Once MutL finds a nick, it triggers the degradation of the nicked strand all the way back through the mismatch.

Base Excision Repair

Base excision repair uses specific DNA glycosylases to recognize and remove damaged bases.
DNA glycosylases recognize specific lesions, cleave the N-glycosidic bond, and create an apurinic/apyrimidinic (AP) site.
Examples of DNA glycosylases include uracil glycosylase, which removes uracil from DNA, and other glycosylases that create AP sites at 8-hydroxyG, hypoxanthine, and 3-methyladenine.

Repair at AP Sites in Bacteria

The entire nucleotide is ultimately removed, not just the damaged base.
AP endonucleases cut the DNA backbone around the AP site, making a nick in the DNA.
DNA Pol I synthesizes new DNA, and DNA ligase seals the nick.

Base-Excision Repair (Recap)

A DNA glycosylase recognizes a damaged base and cleaves between the base and deoxyribose in the backbone.
An AP endonuclease cleaves the phosphodiester backbone near the AP site.
DNA polymerase I initiates repair synthesis from the free 3′ hydroxyl at the nick, removing and replacing a portion of the damaged strand.
The nick remaining after DNA polymerase I has dissociated is sealed by DNA ligase.

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