Podcast
Questions and Answers
What is the core sequence of Chi sites in enteric bacteria such as E. coli and Salmonella?
What is the core sequence of Chi sites in enteric bacteria such as E. coli and Salmonella?
- 5'-GCTGATGG-3’
- 5'-GCTGGTAG-3’
- 5'-GCTGGTGG-3’ (correct)
- 5'-GCTGCTGG-3’
What function does the N-terminal domain of the RecB protein serve?
What function does the N-terminal domain of the RecB protein serve?
- Dimerization
- DNA recognition
- Nuclease activity
- Helicase function (correct)
Which key catalytic residues are found in the nuclease motif of RecB?
Which key catalytic residues are found in the nuclease motif of RecB?
- Serine and threonine
- Aspartate and lysine (correct)
- Cysteine and histidine
- Arginine and glutamate
Which protein is responsible for recognizing the Chi site and how many amino acids does it contain?
Which protein is responsible for recognizing the Chi site and how many amino acids does it contain?
Which of the following motifs are characteristic of the SF1 helicase found in the RecB protein?
Which of the following motifs are characteristic of the SF1 helicase found in the RecB protein?
What is the first step in the process of homologous recombination?
What is the first step in the process of homologous recombination?
What structural formation results from strand invasion during homologous recombination?
What structural formation results from strand invasion during homologous recombination?
During which part of homologous recombination is heteroduplex DNA formed?
During which part of homologous recombination is heteroduplex DNA formed?
What happens during the branch migration step of recombination?
What happens during the branch migration step of recombination?
What enzymatic complex processes DNA ends following a double-strand break?
What enzymatic complex processes DNA ends following a double-strand break?
What is the function of RecBCD at a Chi site during recombination?
What is the function of RecBCD at a Chi site during recombination?
How are Holliday junctions typically resolved?
How are Holliday junctions typically resolved?
Chi sites, also known as recombination hotspots, are characterized by which feature?
Chi sites, also known as recombination hotspots, are characterized by which feature?
What is the primary function of the Ku heterodimer in the C-NHEJ pathway?
What is the primary function of the Ku heterodimer in the C-NHEJ pathway?
Which components are essential for forming the DNA-PK holoenzyme in C-NHEJ?
Which components are essential for forming the DNA-PK holoenzyme in C-NHEJ?
Which statement correctly describes the characteristics of the Ku protein?
Which statement correctly describes the characteristics of the Ku protein?
What is the primary role of DNA-PKcs in the C-NHEJ mechanism?
What is the primary role of DNA-PKcs in the C-NHEJ mechanism?
Which of the following enzymes is NOT involved in the processing of DNA ends during C-NHEJ?
Which of the following enzymes is NOT involved in the processing of DNA ends during C-NHEJ?
What distinguishes C-NHEJ from other DNA repair mechanisms regarding DNA ends?
What distinguishes C-NHEJ from other DNA repair mechanisms regarding DNA ends?
What is the role of the LIG4/XRCC4/XLF complex in the C-NHEJ pathway?
What is the role of the LIG4/XRCC4/XLF complex in the C-NHEJ pathway?
Which of these choices is a consequence of Ku's initial binding to DNA ends?
Which of these choices is a consequence of Ku's initial binding to DNA ends?
What characterizes the mechanistic flexibility of the NHEJ process?
What characterizes the mechanistic flexibility of the NHEJ process?
Which statement accurately describes the role of A-NHEJ?
Which statement accurately describes the role of A-NHEJ?
What suppresses the activity of A-EJ?
What suppresses the activity of A-EJ?
What has been proposed about the factors involved in A-EJ?
What has been proposed about the factors involved in A-EJ?
When does A-EJ typically engage in DSB processing?
When does A-EJ typically engage in DSB processing?
What is the relationship between A-EJ and C-NHEJ?
What is the relationship between A-EJ and C-NHEJ?
Which of the following best describes the speed of A-EJ compared to C-NHEJ?
Which of the following best describes the speed of A-EJ compared to C-NHEJ?
What is one of the implications of the functional diversity of A-EJ factors?
What is one of the implications of the functional diversity of A-EJ factors?
In which phase of the cell cycle is the dependency on BRCA1/CtIP/MRN more likely?
In which phase of the cell cycle is the dependency on BRCA1/CtIP/MRN more likely?
What starts Non-Homologous End Joining (NHEJ) in the repair process?
What starts Non-Homologous End Joining (NHEJ) in the repair process?
Which enzyme can trim incompatible DNA ends during NHEJ?
Which enzyme can trim incompatible DNA ends during NHEJ?
After DSB resection in homologous recombination, what is the first protein that coats the single-stranded DNA?
After DSB resection in homologous recombination, what is the first protein that coats the single-stranded DNA?
What is the role of Rad51 in homologous recombination?
What is the role of Rad51 in homologous recombination?
What complex seals the break in Non-Homologous End Joining?
What complex seals the break in Non-Homologous End Joining?
What happens to the break after resection in homologous recombination?
What happens to the break after resection in homologous recombination?
Which protein assists Rad51 in the replacement of RPA during homologous recombination?
Which protein assists Rad51 in the replacement of RPA during homologous recombination?
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Study Notes
Chi Sites and DNA Repair in Bacteria
- Chi sites are unique sequences that stimulate DNA double-strand break (DSB) repair in bacteria.
- In enteric bacteria like E. coli and Salmonella, the core sequence of the Chi site is 5'-GCTGGTGG-3’.
- The Chi site includes an additional 4 to 7 nucleotides on the 3' side, essential for function.
RecBCD Enzyme Structure
- RecBCD enzyme is crucial for processing DSBs in bacteria.
- Comprised of three proteins:
- RecB: 1180 amino acids, features:
- N-terminal domain with seven SF1 helicase motifs.
- C-terminal domain with nuclease motifs containing key aspartate and lysine residues.
- RecC: 1122 amino acids, contains the Chi recognition site.
- RecD: 608 amino acids, also has seven conserved SF1 helicase motifs.
- RecB: 1180 amino acids, features:
Homologous Recombination Process
- Homologous recombination involves three key steps:
- Strand exchange: Pairing of broken DNA with homologous sister chromatid regions.
- Branch migration: Extends regions of heteroduplex DNA formed during crossover.
- Resolution: Cleavage of Holliday junctions to separate duplex DNA.
Key Processes in Homologous Recombination
- Resection creates 3' single-stranded DNA tails necessary for strand invasion.
- Strand invasion uses free 3' ends to form Holliday junctions, initiating DNA synthesis.
- Branch migration translocates the Holliday junction along the DNA, extending heteroduplex areas.
Non-Homologous End Joining (NHEJ) Pathways
-
Canonical Non-Homologous End Joining (C-NHEJ):
- Dependent on Ku70/80 heterodimer and DNA-PK catalytic subunit (DNA-PKcs).
- Ku binds DSBs, providing a docking site for DNA-PKcs and facilitating repair.
- Various nucleases, such as Artemis, process DNA ends for ligation by the LIG4/XRCC4/XLF complex.
-
Alternative End Joining (A-EJ):
- Functions alongside C-NHEJ as a backup repair pathway if standard processes fail.
- Typically engaged at DSBs where C-NHEJ or homologous recombination have been ineffective.
- A-EJ is more prevalent in G2 phase cells due to dependencies on repair factors present.
General Mechanisms of DSB Repair
- Non-homologous end joining begins with Ku recognition of DNA ends.
- End processing by nucleases like Artemis can occur if ends are incompatible.
- Ligase IV complex finalizes the break sealing.
- Homologous recombination requires MRN-CtIP complex to initiate resection and generate ssDNA coated by RPA, later replaced by Rad51 for strand invasion.
Importance of DNA Repair Mechanisms
- Ku proteins are essential for both prokaryotic and eukaryotic genome integrity.
- Multi-faceted repair pathways like C-NHEJ and A-EJ allow cells to adapt to varied DNA break configurations.
- Understanding these processes enhances knowledge of genetic repair mechanisms and potential cancer therapies.
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