The Difference Between Non-Homologous End Joining and Homology Dependent Recombination

A technical article about the process of NHEJ & HDR, and how these two processes differ.

While I was doing research about genomics and CRISPR I came across all these different ways cells repair double stranded breaks (DSB).

Homology directed repair, non-homologous end joining, homologous recombination and the list of processes goes on and on. But I couldn’t find any articles on the difference between each of these systems.

So I decided to make this article for all those people like me who need an article that will go over the different repair processes in a cell.

Overview

The most complicated thing about DSBs is that since both RNA strands are broken, there is no template DNA for the cell to copy. This means that when a DSB occurs, the cell needs to be resourceful and use the resources it has to make a template DNA to fix the break.

The two repair processes that I am going to go over in this article are:

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

HDR and NHEJ are the most common repair process that occur inside cells. In this article I am going to dive deeper into both of these repair processes.

Homology-directed repair (HDR)

HDR is a repair process that invades an undamaged genome with a similar DNA sequence to the broken strand, synthesizing this undamaged DNA to use for the broken strand.

Steps

HDR is activated by a DSB in DNA.

When a DBS first occurs in the genome, MRN complex will come and bind to the end of the genome.

The MRN is a complex made up of three proteins — Mre11, Rad50 and Nbs1. MRN’s main function in cells is to help cells to repair DSB.

Now DNA resection occurs. This means that the MRN will cut the 5' ends of DNA, leaving the 3' end as a blunt overhang.

DNA polymerase now attaches to the MRN

and then the broken DNA attaches itself to the DNA of the neighbouring genome.

The DNA polymerase pulls this neighbouring strand of DNA apart.

As the DNA polymerase is pulling the the DNA apart it synthesizes the upper strand, using the upper strand of DNA as a template for the broken DNA. This is the step of HDR where the DNA is synthesized and then repaired.

As the DNA is synthesized it creates something called a holiday junction, a situation where two DNA strands are intertwined.

There are two different process that the cell can undergo to resolve the holiday junction, so that both strands of DNA separate into their original form. These two processes are called resolution and cleavage.

In the resolution process a protein that cleaves DNA called resolvasome, surrounds the genome.

The resolvasome cleaves the DNA where the two strands overlap.

And now the two strands of DNA swap places and overlap with each other. One strand of the damaged DNA joins one strand of the template DNA; and the other strand of damaged DNA joins the remaining strand of template DNA. The two genomes have swapped RNAs.

Then DNA ligase smooths the break. The DNA is now repaired to it’s pre-break form.

And the other way the DNA can finish repairing itself is through a process called cleavage.

First resolvasomes surround the DNA.

Then the resolvasomes cleave the DNA directly in the middle of the two strands.

But instead of crossing over it’s DNA like in resolution, both genomes collapse back into their original positions.

DNA ligase smooths the break, repairing the DNA back to it’s pre-break form.

And that is the full process of how a double stranded break is repaired with HDR.

Advantages of HDR

• It works very well for controlled modifications.
• Is very accurate.
• Designing and creating a template piece of DNA for HDR is quick and inexpensive.
• A circular or linear plasmid can be used to transport the template DNA into the cell.

Disadvantages of HDR

• Needs a template DNA strand.
• It is a time consuming process.
• It is only 83% efficient.
• HDR causes many off-target effects.

Non-homologous end joining (NHEJ)

NHEJ is the other repair process that occurs most often in cells to repair DSB. NHEJ is the process that repairs DSB caused by CRISPR-cas9 in cells.

NHEJ basically works by jamming the broken DNA back together.

Steps

A DSB activates NHEJ.

Ku70/Ku80 heterodimer binds to the broken ends of DNA. Ku is a protein molecule (heterodimer) which function is to aid the repair process in NHEJ.

Ku recruits DNA DNA-PKcs to help repair the break. DNA-PKcs is a protein kinase that is inactive on it’s own and relies on Ku to activate it so it can use it’s kinase activity on the break. (Kinase activity is an enzyme that phosphorates other enzymes, which means that it helps to join enzymes together.)

DNA-PKcs then recruits DNA Artemis to help repair the break.

Artemis trims any single stranded tails that are present at the break.

Now Ligase4, acting in a complex with XRCC4 and XLF/Cernunnos attaches directly to the broken area pushing Artemis off the genome.

The Ligase4 complex repairs the break by joining both strands of DNA together. Remember that NHEJ doesn’t synthesize the broken DNA, it actually deletes it, pushing both halves of DNA back together and deleting the broken section.

DNA ligase smooths the break.

And now the DNA is fully repaired. *Remember that the DNA is not repaired to it’s pre-break form, since a large chunk of DNA was deleted in the process.*

Advantages of NHEJ

• Doesn’t need a template DNA strand.
• Has a higher capacity for repair than HDR.
• It is active throughout the whole cell.
• Creates accurate deletions and insertions.
• Useful for gene knockout

Disadvantages of NHEJ

• It is very error prone.
• Has many off-target effects.
• Some DNA bases will always be lost when NHEJ occurs since no synthesis aids the break.

And that, my friends, wraps up this article about NHEJ and HDR. Hopefully this made everything more clear about the different repair processes that occur inside cells to repair double stranded breaks.

If you want to dive deeper into genome repair processes, check out some of these awesome resources🔥:

• Great animation of non-homologous end joining
• Homology directed repair article
• Non-homologous end joining article
• Talk about characterizing large HDR insertions

And that’s it for now! See you all next time!

My name is Rachel, I am a grade 10 student who’s main goal in life is to end poverty and hunger. I also love being outside, spending time with my family, and learning about science and technology. If you have any suggestions, questions or just want to talk, you can email me at: runnerrachel.lee@gmail.com, or message me on LinkedIn. Don’t forget to sign up for my monthly newsletter here. Thanks so much for reading!