MCB 250 – Homologous Recombination and Non-homologous End Joining

Module 20 (VCasts 43-44): Describe the general steps in the RecBCD pathway of homologous recombination | Describe the functions of each of the major proteins/complexes involved in the process: RecBCD, RecA, RuvAB, and RuvC | Describe the relationship between strand invasion and repair/DNA synthesis versus Holliday junction migration and resolution | Define the difference between a crossover and a patch, resulting from recombinational repair, and the relationship to RuvC | Describe the primary role of recombinational repair in E. coli and the general role in meiosis in eukaryotes | Describe the general steps in non-homologous end-joining (NHEJ) | Compare and contrast homologous recombination with NHEJ | Describe the overall use of CRISPR and the relationship with homologous recombination versus NHEJ

what is the most common problem repaired by recombination?

collapsed replication fork

how does replication fork collapse?

nick in front of replication fork → single-stranded break

how can we repair collapsed replication fork?

homologous recombination

how can we repair double stranded breaks in DNA?

homologous recombination

what are double-stranded breaks caused by?

• oxidative damage

• radiation damage

• cellular nucleases

what is homologous recombination?

a DNA rearrangement → DNA is cut and rejoined

why is homologous recombination important?

• to repair DNA

• required for meiosis

  • for proper chromosome segregation

  • for generating diversity in gametes (crossing-over)

• allows genes to be transmitted from one bacterial strain to a closely related strain

what is the primary role of homologous recombination?

to repair DNA

which organisms does recombination occur in?

all organisms: viruses, bacteria, archaea, eukaryotes

how can we tell if there is a double stranded break in DNA?

if there is a clear 3’ end and 5’ end

explanation: bacteria is a circle → there are no ends; eukaryotes have telomeres that are attached to the nuclear envelope → not a clear 3’ or 5’ end

what is used as a template for recombination?

a homologous DNA

overall steps of homologous recombination

1. process DNA to generate 3’ overhangs

2. 3’ strand invasion at homologous DNA sequence → Pol I synthesizes new DNA and fills in gaps

3. branch migration → generation of two holliday junctions

what can holliday junctions do?

migrate down DNA → move crossover region AWAY from region that needs to be repaired → this stabilizes the interactions between the two pieces of DNA

can branch migration occur if there is a difference in base pair between the two homologous DNA?

yes → this base pair mismatch can be repaired via mismatch repair

how can a holliday junction be resolved?

• vertical DNA cleavage → crossover

• horizontal DNA cleavage → patch

what are the chances for a crossover to occur as a result of a holliday junction resolution?

50%

is branch migration (movement of holliday junction) independent or dependent of DNA synthesis?

independent

what are the enzymes responsible for homologous recombination in e. coli?

for the primary pathway (RecBCD pathway):

• RecA

• RecBCD

• RuvABC

• ss-binding protein, Pol I, ligase

what is the primary pathway for homologous recombination called?

RecBCD pathway

purpose of RecBCD

generates a 3’ overhang to allow strand invasion

what is RecBCD?

a complex that consists of 1 helicase and 2 nucleases

what is the substrate for RecBCD?

ds-break

steps that RecBCD goes through

1. helicase unwinds/separates the 2 DNA strands

2. nuclease degrades the 2 DNA strands

3. continues degrading until it reaches the chi (χ) site (a particular 8 base sequence in the DNA)

4. degradation of 5’ end continues; degradation of 3’ end slows down → creates 3’ overhang

what is the chi site?

a particular sequence in DNA that RecBCD recognizes; signal for RecBCD to stop degrading 3’ end

how often are chi sites represented in e. coli genome?

1/4.5 kb → they are significantly overrepresented (predicted is 1/65 kb)

what is the substrate for RecA?

the 3’ overhang that RecBCD created

what does RecA do?

1. polymerizes 5’→3’ on the 3’ overhang

2. searches the entire chromosome (all DNA in cell) for the homologous sequence

3. initiates strand invasion → creates duplex formation (holliday junction)

how many base pairs does RecA need to search for homology? how many base pairs does it prefer?

requires at least 50 bp; works better with longer regions (~500 bp)

is holliday junction independent or dependent of RecA?

independent

what is branch migration (of holliday junction) mediated by?

RuvAB

what is the substrate for RuvAB?

holliday junctions

what is the main purpose of RuvAB?

mediates branch migration of holliday junctions using RuvB and RuvA proteins

what is RuvB and what does it do?

an ATP-dependent helicase part of the RuvAB complex that uses ATP to spool DNA through RuvA protein

what is RuvA and what does it do?

a protein part of the RuvAB complex that recognizes and binds to the holliday junction

what does RuvC do?

cleaves the holliday junction → random orientation → results in either crossover or patch

can homologous recombination repair a ds-break perfectly?

yes

can non-homologous end joining repair a ds-break perfectly?

no

what does homologous recombination use and for what?

uses information in an intact ds-DNA molecule to repair the broken DNA molecule

does non-homologous end joining need a copy of DNA to repair?

no, it does not require a template

why can’t homologous recombination be used to fix a ds-break in eukaryotes?

Because homologous recombination requires a homologous template, which may not be available during certain stages of the cell cycle → only available during S and G2

what is the main mechanism for ds-break repair in eukaryotes?

non-homologous end joining

what does non-homologous end joining do and how?

repairs ds-breaks by trimming and joining the ends of linear, ds-DNA molecules

basic steps of non-homologous end joining

1. protein (Ku) recognizes the ds-break

2. protein kinase (DNA-PKcs) recruits nuclease (Artemis)

3. nuclease (Artemis) processes the ends

4. DNA ligase seals/joins the ends

what protects chromosome ends from non-homologous end joining and how?

telomeres are coated with proteins and ∴ not recognized by the non-homologous end joining machinery

what type of dna repair do bacteria use?

homologous recombination

what can simultaneous ds-breaks lead to?

translocations → swapped pieces of DNA from separate chromosomes → can lead to death or different expression of genes

where is crispr/cas9 system found in?

bacteria and archaea

what does crispr/cas9 system do?

uses RNA to target DNA to create ds-breaks

which type of breaks does non-homologous end joining recognize?

internal ds-breaks in DNA

what is simultaneous ds-breaks caused by?

non-homologous end joining