Set 6: DNA replication, repair proteins, and types of DNA repair

Why is DNA replication and repair important in CRISPR?

CRISPR immunity and editing depend on:

• DNA replication machienery

• DNA repair pathways

• recombination systems

CRISPR does not act in isolation - it is embedded within fundamental genome maintenance in DNA replication.

Describe bacterial cell replication, and how it is utopian?

Replication is bi-directional, it goes both ways. The replication begins at the origin, where a ‘replisome’ machine assembles.

The replisome machine synthesises new DNA semi-conservitavley, each new DNA molecule consists of one original parental strand and one newly synthesised daughter strand.

Smooth genome duplication often fails, as it is a complicated machine and consequently susceptible to DNA damage and breakdown under stress. It can break due to DNA strand breaks, protein obsticals (such as transcription complexes or RNA pol), DNA-DNA cross links, DNA-protein cross links.

This stops helicases and polymerases running along DNA, leading to chromosome fragmentation, and required repair to resume replication.

What is in the replisome machinery?

Helicase (DnaB) - ring shaped motor protein that unwinds DNA double helix using energy from ATP hydrolysis.

DNA pol III - enzyme that synthesises new DNA strand (one continuously and one lagging (okazaki)).

Primase (DnaG) - lays RNA primers that allow DNA polymerase to begin synthesis.

Sliding clamp (PCNA) - ring that keeps DNA pol attached to DNA, ensuring high processivity (speed).

Clamp loader (RFC) - loads sliding clamp to DNA.

Single-strand binding protein (SSB) - stabilises unwound ssDNA to prevent it from re-annealing or forming secondary structures.

What does DNA repair do and why did it evolve?

DNA repair fixes broken genome duplication. Homologous recombination evolved to support DNA replication, it had to evolve to do so or replication that is broken would never be fixed - life on earth would end.

1% (~50,000 nts) of bacterial genome has gaps leading to improper replication, under stress e.g UV exposure, this increases to ½ a million nts.

Post replication gap repair fills in the gaps via homologous recombination.

How does bacterial cell generate immunity to a virus before virus kills the cell? How was this discovered?

It does this through DNA repair and replication - Cas1/Cas2 can recognise replication forks that need repair and targets it.

This was discovered in a study whose hypothesis: cells encountering defective replicating phage develop rapid immunity, survive, and form the surviving population, called DNA replication-repair triggered immunity.

This research found: UV irradiation used to pre-damage virus DNA, damaged virus DNA into bacteria, triggers replication with high levels of stalling, this leads to much higher rates of CRISPR adaptation in so called 'bacteriophage insensitive mutants' (BIMs).

What is Rec BCD?

a 330-kDa, three-subunit enzyme complex (RecA, RecB, RecC) in Escherichia coli that acts as a highly processive helicase and nuclease to repair DNA double-strand breaks (DSBs) via homologous recombination.

• This senses gaps, processes dsDNA breaks and repairs them by loading RecA onto DNA.

What is RecA?

A repair protein that functions by forming a helical filament on single-stranded DNA to facilitate the search for, and exchange of genetic material.

What is RecFOR?

A repair protein responsible for initiating DNA damage repair by facilitating the loading of the RecA recombinase onto single-stranded DNA

Experimental evidence for activity of Cas1-Cas2 during DNA replication?

• Research shows if we fuse Cas1-Cas2 to yellow fluorescent tag when DNA replication is occurring (Cas1-yfp-Cas2 fully active), we see 2 yellow blobs per cell representing 2 replisomes going around cell DNA in replication (bidirectional replication).

• If we inactivate replication at 42 degrees C, protein becomes unstable, DNA replication cannot occur, and fluorescence disappears;

How does RecBCD work with Cas1-Cas2 in DNA repair? Experimental evidence?

RecBCD interacts physically with Cas1-Cas2, when this occurs Cas1-Cas2 allosterically 'tames' it.

This prevents RecBCD from going too fast and repairing things too quickly because if it does go too fast, there is no substrate for Cas1-Cas2.

Experimental evidence found:

RecBCD (50nM) nuclease-helicase activity is inhibited by Cas1-Cas2 (200mM) in FRET assays, consistent with genetic evidence for DNA repair at replication forks stimulating CRISPR immunity.

We predict that RecBCD is controlled by Cas1-Cas2 to stimulate CRISPR immunity, inhibit recombination, and interact physically with RecB of the RecBCD complex.

What is CRISPR-Cas immunity evolved from?

DNA replication repair.

What are the 5 different types of DNA repair?

DNA cutting

Base editing

DNA integration

Prime editing

DNA nicking

Describe DNA cutting as DNA repair?

This uses Cas9/Cas12 nuclease activity to cut DNA creating a double strand break.

This is repaired using End joining for; deletion, insertion or substitution.

or

HDR for: precise deletion, precise insertion or precise substitution.

Describe base editing as DNA repair?

This uses daminase or glycosylase activity, and results in a deaminated nucleotide of abasic site.

DNA repair is done by MMR(mismatch repair)/SSBR(single-strand base repair) for changing C to T or A to G.

or

TLS (transversion substitution) to convert C to G

or

BER (base excision repair) for precise deletion.

Describe DNA integration as DNA repair?

This uses recombinase transposase to cut out an integration intermediate.

This is repaired using transposition to insert sequence in-between inverted repeats,

or

Site-specific recombination where desired insertion is inserted between recombinase sites

Describe Prime editing as DNA repair?

This uses reverse transcriptase, and creates a flap structure which is removed via flap excision in order to create a precise substitution, deletion or insertion.

Describe DNA nicking as DNA repair?

This uses Cas9 nickase to create a single strand break.

Repair is done using SSBR for a deletion, insertion or substitution,

or

HDR for a precise deletion, insertion or substitution.

What are the main class 2 CRISPR system interference enzymes used in biotechnology?

Cas9, Cas12, Cas14, CasX