New & Noteworthy

Look Before You Leap…Into DNA Repair

June 09, 2016

It is worth a pause before leaping into action. This is true at the edge of a cliff, making a life or death situation, and before DNA repair. Cliff jumping image from Evan Bench on flickr, emergency technician image from Wikimedia Commons

Sometimes in an emergency, it can be useful to take a pause before trying to fix a problem. If the ambulance is on the way you may not want to start removing someone’s appendix right then and there. Yes you may save them, but the ambulance and EMTs will do less damage to the patient once they get there.

The same sort of logic applies to cells too. For example, a double stranded break is a deadly emergency that can wreak havoc with a cell’s genome.

The cell can deal with this in a few ways; the big two being non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ is the simplest in that the broken ends are simply stuck back together. However, it often results in a bit of DNA damage as a few bases are added or lost at the ligation point.

The other option, homologous recombination (HR), involves using a homologous DNA region as a template to essentially resynthesize the broken area. There are two possible ways this can happen in a cell: gene conversion and break induced replication (BIR).

If both ends are available without too much of a gap and in the right orientation, the cell opts for gene conversion, the safer of the two options. Only a small section of DNA is resynthesized, keeping most of the original DNA intact.

It is a different story for BIR, the second approach. Here, a large swath of DNA gets resynthesized, meaning a big loss of heterozygosity (LOH)—two sections of DNA are now identical over a large region. BIR only happens when there is only one end available or when there is a big gap of missing DNA.

Of the two HR possibilities, gene conversion is preferable over BIR. This is why the cell wisely waits to make sure there is no other option before starting down this path. This pause goes by the name of recombination execution checkpoint (REC).

A new study by Jain and coworkers out in GENETICS has identified two of the key players involved in this checkpoint—SGS1 and MPH1. Both are highly conserved 3’ to 5’ helicases.

These researchers found that when both are deleted, the REC disappears. And that isn’t all. In situations where the wild type cell would choose to repair its DNA by BIR, the double deletion strain no longer does. Instead, it now uses a process that is more similar to gene conversion.

Jain and coworkers found this using a reporter that contained an HO endonuclease site in the middle of the LEU2 gene. Once the HO endonuclease is activated, it makes a double strand DNA break, cutting the LEU2 gene in half.

The cell was provided with a variety of templates to be used in HR. One of these only provides the part of the LEU2 gene to the right of the HO endonuclease site. As only one of the ends has homology to the cleaved LEU2 gene, with this template, the cell can only use BIR to repair the break.

The researchers saw a huge increase in how fast the DNA was repaired using the BIR-only template in the strain deleted for both sgs1 and mph1 compared to the wild type strain. The double mutant repaired the DNA using BIR nearly as quickly as a wild type cell could using gene conversion. The pause before repair was essentially lost in the double mutant.

The double deletion strain wasn’t just faster with the BIR-specific template either. It repaired DNA via this pathway about 4 times better than the wild type strain did.

There are at least a couple of different reasons why the double mutant could be so much better at repairing DNA in BIR situations. In the first, the cell is just better at BIR—it can initiate the BIR pathway much more quickly than wild type. The second possibility is that this double mutant isn’t using the BIR pathway anymore and is using something closer to gene conversion.

Jain and coworkers found that the second option is the more likely of the two. The way they figured this out was to make a mutation in the POL30 gene, a gene required for DNA repair by BIR but not gene conversion. So, they tested what happens when POL30 is mutated in the wild type and the sgs1 mph1 double deletion strains.

They found that while a dominant negative mutant of pol30 had the predicted effect of severely compromising repair in wild type cells using their BIR-specific reporter, it had no effect in the double deletion mutant. Since Pol30p is needed for the BIR pathway, the strain deleted for sgs1 and mph1 must be using a different pathway to fix the DNA damage. So the pause is eliminated because the cell isn’t really using the BIR pathway anymore.

Image from The Petrick on vimeo

We don’t have time to go into it here, but there is a lot more research in this paper that looks at why the REC might be lost and that probes the differences in how MPH1 and SGS1 influence the BIR pathway that I encourage you to read. For example, deleting SGS1 is not identical to deleting MPH1 in terms of the BIR DNA repair pathway. And each single mutant still uses the BIR DNA pathway as the pol30 mutation severely compromises the ability of each to repair the BIR-specific reporter.

There is a lot more fascinating stuff like this in the study. The bottom line, though, is that MPH1 and SGS1 are the level-headed people urging folks not to panic and to wait for the EMTs to get to the accident scene to help. When MPH1 and SGS1 are gone, the cell dives right in and starts repairing breaks without waiting for the safest option—gene conversion. Who knows what havoc is wreaked in their absence!

by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight