Finding the missing link in plant DNA damage response
DNA defines how proteins are produced and how all the processes inside the cell occur. For this reason, when internal or external factors cause DNA damage, it is imperative to quickly detect and repair this damage to preserve genome integrity. DNA damage triggers an alarm state called DNA damage response (DDR). DDR depends on the phosphorylation of histone H2A.X, which acts as an alarm beacon that guides other proteins to the site of DNA damage and allows them to mount a fast and effective response to repair the DNA damage. In mammals DDR, H2A.X is recognized by the protein MDC1 through a tandem BRCT (tBRCT) domain. MDC1 is then able to recruit other DDR proteins, which are the effectors for the different DNA repair mechanisms.
DDR is a highly conserved mechanism throughout evolution, and scientists have been able to identify DDR proteins in plants that have homology and similar functions to those of mammals. However, identifying a homolog of the MDC1 protein had remained elusive until now. In an article published on EMBO Reports on March 4th, 2024, the group of Frédéric Berger, reports the identification of protein BCP4 as the functional counterpart of MDC1 in plants.
So far, the existence of a plant MDC1 counterpart had been difficult to prove. “We were all looking at it in the wrong way”, explains Zdravko Lorković, first author of the work and senior researcher in the lab of Frédéric Berger. “Everyone was looking at human DDR proteins and trying to find a similar protein in plants, which is normally a good strategy but didn’t work in this case”.
Indeed, Lorković utilized a different approach to crack this particular nut. “We first set out to identify all plant proteins that contained BRCT domains and then performed phylogenetic analysis. One of these proteins, BCP4, clustered together with human MDC1”, Lorković explains. BCP4 protein differs greatly from mammalian MDC1, missing all functionally important sequence motifs of MDC1 apart from tBRCT. However, the team observed that the three-dimensional structure of their respective tBRCT domains was very similar. “Using AlphaFold-based predictions, we showed that only the tBRCT domain of BCP4 can recognize phosphorylated H2A.X,” explains Lorkovic. To confirm whether BCP4 was a functional counterpart to MDC1, the team used a combination of genetics, biochemistry, and imaging techniques. “We saw that BCP4 bound phosphorylated H2A.X, co-localized with H2A.X during DNA damage, and that BCP4 mutants were sensitive to DNA damage because DDR was not working,” says Lorković.
To further validate the function of BCP4, the group studied which proteins can bind to BCP4 and be recruited to the DNA damage site. “We have evidence that BCP4 binds to the MRN complex, a main DDR effector which is also recruited by MDC1 in mammals,” explains Lorković. Interestingly, the protein domain of BCP4 that binds MRN is completely different from that of MDC1, which highlights that some ancient proteins may have evolved very differently in plants and animals but still retain the same core function.
Further dissection of the interactions and function of BCP4 and other proteins in the plant DDR pathway will help better understand this crucial process.