DNA damage recognition: keeping the right players on board

DNA breaks are an unfortunate consequence of the biochemical processes that keep our cells alive. Thankfully, we are well equipped to deal with these potentially lethal changes with a cascade of DNA damage recognition and repair mechanisms that kick in when a break is detected.

Genomic DNA is continually being damaged. This figure shows the types of damage that can occur

Genomic DNA is continually being damaged. This figure shows the types of damage that can occur

Dr Nick Lakin and his group, in collaboration with colleague Dr Catherine Pears, have recently published a paper that describes some of the intricate processes that facilitate repair of DNA breaks (1). Their work has implications for cancer research, where there is a strong interest in developing treatments to exploit the weakness in DNA damage repair that cancer cells frequently show.

The two researchers use Dictyostelium discoideum, a simple haploid eukaryote, to study DNA repair pathways. Dictyostelium is an attractive model system because it can be manipulated genetically and has DNA repair enzymes similar to those found in humans. These enzymes are equipped to repair both DNA single-strand breaks and double-strand breaks (DSBs).

Lifecycle of Dictyostelium discoideum(Strmecki et al (2005). Dev Biol. 284, 25-36)

Lifecycle of Dictyostelium discoideum(Strmecki et al (2005). Dev Biol. 284, 25-36)
(Click to enlarge)

‘We are interested in enzymes called PARPs which function in signalling DNA damage,’ explains Dr Lakin. ‘Their role is best defined in the repair of single-strand DNA breaks. They are also part of an early response to DSBs, but nobody knows what they do here.’

PARPs [poly ADP-ribose (PAR) polymerases] are recruited to the site of damage and catalyse addition of a polymer of ADP-ribose unit onto proteins, Their targets are not known, but PARylation, as this addition is known, is thought to relax the chromatin structure around the break, possibly enabling access of repair proteins to the site of damage. The polymers also provide interaction domains for other proteins, a process that is likely to be important for recruiting and retaining repair components at sites of DNA damage.

There are around 12 PARPs in Dictyoselium. Research from Dr Lakin and colleagues revealed one, PARP1a, which, unlike the others, is not required for single-strand breaks but is essential for PARylation at DSBs.

Since double-strand DNA repair can take place in two ways in the cell – through homologous recombination (HR) and non-homologous end joining (NHEJ) - the next question for the researchers was which pathway PARP1a was working in.

PARylation at sites of DNA damage in Dictyostelium cells. Cells were treated with an agent that induces DNA single-strand breaks (H<sub>2</sub>O<sub>2</sub>) as indicated. PARylation of proteins is represented by the formation of DNA damage induced foci in nuclei visualised by immunofluorescence using antibodies that specifically recognise PAR chains. Nuclei are represented by staining with DAPI

PARylation at sites of DNA damage in Dictyostelium cells. Cells were treated with an agent that induces DNA single-strand breaks (H2O2) as indicated. PARylation of proteins is represented by the formation of DNA damage induced foci in nuclei visualised by immunofluorescence using antibodies that specifically recognise PAR chains. Nuclei are represented by staining with DAPI (Click to enlarge)

To shed light on this, the group knocked out the gene for PARP1a in Dictyostelium and investigated how this affected the ability of cells to perform DSB repair. The results indicated that the enzyme promoted the NHEJ pathway at the expense of the HR pathway.

The researchers turned next to a well-characterised component of the DNA repair process called Ku, which avidly binds to DNA ends and favours the NHEJ pathway. ‘Ku is one of factors known to govern the choice between the two pathways,’ says Dr Lakin, ‘so we used it to look at the molecular basis of PARP1a function.’

The researchers found that PARP1a facilitated the retention of Ku at the break site, explaining how the enzyme may be acting to promote NHEJ. Further evidence of Ku’s involvement came from the discovery that Ku protein carries a motif that is known to bind to PAR and that this motif is required for retention of Ku at DSBs and efficient NHEJ.

These results have led Dr Lakin to propose how Ku and PARP1a may be working together to promote NEHJ. ‘Our model is that PARP1a recognises the break, becomes activated, catalyses the PARylation event on specific targets, and that this somehow enables Ku to be retained to promote repair.’ He believes that PARP1a involvement is likely to be changing the dynamics of the proteins at the break site to keep hold of Ku, so that repair complexes can do their job.

Together with a paper from another group published at the same time describing a similar process in human cells, this is the first demonstration that PARylation plays a role in NHEJ and an explanation for how this happens on a molecular basis.

From a biomedical perspective, the work has implications for the development of cancer drugs. PARPs are a target for treatment for breast cancer, with PARP inhibitors currently targeted at breast cancers which carry mutations in BRCA1 and BRCA2, two cancer susceptibility genes.

The drugs are not yet in the clinic but look very promising in clinical trials. They are thought to work by a synthetic lethal pathway - when a combination of defects in multiple genes leads to cell death whereas a defect in any single gene is viable.

BRCA genes are needed for repair of DSBs by HR. Patients with BRCA1 or BRCA2 mutations are heterozygous for the gene. When a tumour cell loses its functioning BRCA gene, it is unable to repair DNA breaks via HR. PARP inhibitors then impair single-strand break repair, increasing the frequency of single strand breaks being converted to DSBs during DNA replication. The inability of BRCA-defective tumours to rectify this damage by HR kills these cells.

This synthetic lethal approach could be developed to treat other types of cancers, says Dr Lakin. ‘There are lots of cancer genes that are known to affect DNA repair, and people are keen to try and develop inhibitors against these different repair pathways.’

By trying to establish what the different PARPs do in different repair pathways, it may be possible to develop more targeted strategies for treatment.

Dr Lakin is hoping that future work using Dictyostelium will genetically tease out further components of the DNA damage recognition pathways, adding to our knowledge about the function of these processes in health and disease.

References

  1. Couto, C. A., Wang, H.-Y., Green, J.C.A., Kiely, R., Siddaway, R., Borer, C., Pears. C.J. and Lakin, N.D. (2011). PARP regulates non-homologous end-joining through retention of Ku at double strand breaks. J. Cell Biol. 194:367-375.
  2. Pears C.J., Couto C.A., Wang H.-Y., Borer C., Kiely R and Lakin ND (2012).The role of ADP-ribosylation in regulating DNA double-strand break repair.Cell Cycle. 11: 48-56.





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