CORDIS - EU research results

Under the hood: Single-molecule studies of the DNA replication machinery

Final Report Summary - SINGLEREPLISOME (Under the hood: Single-molecule studies of the DNA replication machinery)

The replication of DNA plays a central role in transmitting hereditary information from cell to cell. Our DNA is replicated by a complex, microscopically small machine consisting of several proteins, which all have different tasks but work closely together. One of the proteins unzips the double-stranded DNA, revealing two single strands. Those single strands are then each transformed into a double strand again, resulting now in two identical copies of the original DNA. In this project, we have developed technology to visualise the proteins in the replication machine at the level of single molecules. In particular, we have obtained evidence that the proteins in the replication machinery are continuously being replaced by proteins that are freely diffusing in the cell. This finding is significant because it significantly changes our textbook picture of the replication complex as a stably assembled protein machine. One can compare this with a racing car that is able to replace components, such as its wheels and engine, while racing along the track! This plasticity at the protein level has significant implications for our understanding of how the replication machine deals with roadblocks in the DNA that could give rise to errors and trigger disease.

Further, we have visualised how bacteria deal with damage in their DNA. We have developed tools to image individual proteins inside living cells and have shown that certain DNA copier enzymes related to repair are produced in response to DNA damage and stored in different places in the cell depending on where and when they are needed. This work allowed us to develop microfluidic technology that has immediate applications in health care: we designed a microscopically small device that allows us to visualise how bacteria respond to antibiotics. Not only will these devices help us understand the basic mechanisms of DNA repair and antibiotic-resistance pathways, these devices also may find applications in medical diagnostics.