DNA Repair & Recombination I

Flashcards covering key vocabulary related to DNA repair and recombination mechanisms as discussed in Lecture 17.

What is homologous recombination?

A process by which two DNA molecules exchange genetic information through break and rejoin mechanisms, promoting genetic diversity.

What is the minimum size of homology regions for effective homologous recombination?

The minimum size is typically around 50 base pairs (bp) for efficient repair or integration, though optimal lengths for robust recombination are often several hundred bp or more.

What are two outcomes from resolution of Holliday junctions and how does X-X or X-Y resolution impact the outcome of recombination?

Holliday junction resolution yields patch (non-crossover) or splice (crossover) products.

X-X cleavage leads to non-crossover,

while X-Y cleavage results in crossover.

What is the difference between resolution and dissolution of the Holliday junction, and where can dissolution occur?

Resolution is sequence-specific cleavage by resolvases, producing crossover or non-crossover products.

Dissolution is mediated by helicases (e.g., BLM/Sgs1 in eukaryotes, RecG/RuvAB in prokaryotes) and topoisomerases.

What are the two primary intended outcomes of CRISPR/Cas9-induced double-stranded breaks (DSBs)?

Non-homologous end joining (NHEJ) and Homology-directed repair (HDR).

How does Non-Homologous End Joining (NHEJ) help researchers in genome editing?

NHEJ is an error-prone repair pathway that often results in small insertions or deletions (indels), disrupting gene function and leading to gene knockouts.

How does Homology-Directed Repair (HDR) help researchers in genome editing?

HDR is an accurate repair pathway that uses a provided homologous DNA template to insert or replace specific DNA sequences, enabling precise gene knock-ins.

Which CRISPR/Cas9-induced DNA repair pathway leads to gene knockout, and which leads to gene knock-in?

NHEJ primarily leads to gene knockout, while HDR enables gene knock-in.

What is CRISPR naturally, and what is its origin?

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a natural adaptive immune system found in bacteria and archaea. Its origin lies in its function as a defense mechanism to identify and cleave the DNA of invading viruses and plasmids

What is site-specific recombination?

type of genetic recombination in which DNA strands are broken and rejoined at specific recognition sequences, typically involving specialized proteins (recombinases) that mediate the exchange of DNA segments.

What is an example of site-specific recombination?

The integration of bacteriophage lambda (λ phage) DNA into the E. coli chromosome.

How does site-specific recombination (roughly) work?

It involves specialized proteins (recombinases) breaking and rejoining DNA strands at short, specific recognition sites.

What is the difference between serine recombinases and tyrosine recombinases in terms of their major steps or processes?

Serine recombinases perform a double-strand break, cleaving all four DNA strands simultaneously, and form covalent phosphoserine intermediates, followed by a 180^ ext{o} rotation of DNA segments.

Tyrosine recombinases cleave DNA strands sequentially (typically two at a time), form covalent phosphotyrosine intermediates, and involve Holliday junction-like intermediates during strand exchange.

What are the summary points regarding the major steps and processes of Serine and Tyrosine Recombinases in Site-Specific Recombination (SSR)?

• Serine Recombinases: - Perform a double-strand break, cleaving all four DNA strands simultaneously. - Form covalent phosphoserine intermediates. - Involve a 180^\text{o} rotation of DNA segments.

• - Tyrosine Recombinases: - Cleave DNA strands sequentially (typically two at a time). - Form covalent phosphotyrosine intermediates. - Involve Holliday junction-like intermediates during strand exchange.

What is Spo11?

Spo11 is a topoisomerase-like protein essential for initiating meiotic homologous recombination by creating programmed DNA double-strand breaks.

What are RecBCD and RecA?

RecBCD is a bacterial enzyme complex that initiates homologous recombination by unwinding DNA, creating single-stranded overhangs, and having nuclease activity. RecA is a bacterial protein critical for homologous recombination, catalyzing strand exchange between homologous DNA molecules.

What is a \chi site?

A \chi site (crossover hotspot instigator) is a short DNA sequence (typically GCTGGTGG) that, in E. coli, acts as a recombination hotspot, significantly enhancing the activity of the RecBCD enzyme complex to promote homologous recombination.

What is a Holliday junction?

A Holliday junction is a branched nucleic acid structure formed during homologous recombination, composed of four double-stranded DNA arms joined in a cross-shaped configuration.

What are RuvA, RuvB, and RuvC?

In E. coli, RuvA recognizes and binds to Holliday junctions.

RuvB is a helicase that drives branch migration, actively moving the Holliday junction.

RuvC is a resolvase (endonuclease) that cleaves Holliday junctions, resolving them into recombiant products

What is homologous recombination?

A process by which two DNA molecules exchange genetic information through break and rejoin mechanisms, promoting genetic diversity.

What is illegitimate recombination?

An example of illegitimate recombination is the integration of a retrovirus into a host genome at a random, non-homologous site, which can lead to genomic rearrangements.

What is site-specific recombination?

type of genetic recombination in which DNA strands are broken and rejoined at specific recognition sequences, typically involving specialized proteins (recombinases) that mediate the exchange of DNA segments.

What is λ-phage and the basic principle of its 'life' cycle and integration?

λ-phage is an E. coli bacteriophage with lytic (replication/lysis) and lysogenic (integration as prophage) cycles. Its DNA integrates into the host via site-specific recombination mediated by the λ integrase (Int) enzyme.